BMP-1 Procollagen C-Proteinase for Diagnosis and Treatment of Bone and Soft Tissue Defects and Disorders

ABSTRACT

Uses of BMP-1 isoforms for diagnosing and treating defects and disorders of bone and soft tissues are described. Also described is a newly isolated variant of the BMP-1 isoform BMP-1-3.

RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No. 13/781,070filed Feb. 28, 2013 (in issue), which is a divisional application ofU.S. Ser. No. 12/964,284, filed Dec. 9, 2010 (abandoned), which is adivisional application of U.S. Ser. No. 12/309,510, filed Jan. 21, 2009(now U.S. Pat. No. 7,850,964 B2), which is a United States nationalstage filing under 35 U.S.C. §371 of international application No.PCT/US2007/016605, filed Jul. 23, 2007, designating the U.S., whichclaims priority to U.S. Provisional Application No. 60/832,325, filedJul. 21, 2006.

FIELD OF THE INVENTION

This invention is in the field of diagnosis and regeneration of tissuedefects and disorders. In particular, the invention providescompositions and methods comprising isoforms of BMP-1 to diagnose andtreat tissue defects and disorders.

BACKGROUND

Bone morphogenetic proteins (BMPs) are bone-inducing (osteogenic,osteoinductive) molecules that have been purified and characterized frombone (Sampath and Reddi, Proc. Natl. Acad. Sci. USA, 78: 7599 (1981)).The term “bone morphogenetic protein”, “BMP”, and “morphogen” aresynonymous and refer to members of a particular subclass (i.e., the BMPfamily) of the transforming growth factor-β (TGF-β) superfamily ofproteins (see, e.g., Hoffmann et al., Appl. Microbiol. Biotechnol., 57:294-308 (2001); Reddi, J. Bone Joint Surg., 83-A (Supp. 1): S1-S6(2001); U.S. Pat. Nos. 4,968,590; 5,011,691; 5,674,844; 6,333,312). Allsuch BMPs have a signal peptide, prodomain, and a carboxy-terminal(mature) domain. The carboxy-terminal domain is the mature form of theBMP monomer and contains a highly conserved region characterized byseven cysteines that form a cysteine knot (see, Griffith et al., Proc.Natl. Acad. Sci. USA, 93: 878-883 (1996)). BMPs were originally isolatedfrom mammalian bone using protein purification methods (see, e.g., Uristet al., Proc. Soc. Exp. Biol. Med., 173: 194-199 (1983); Urist et al.,Proc. Natl. Acad. Sci. USA, 81: 371-375 (1987); Sampath et al., Proc.Natl. Acad. Sci. USA, 84: 7109-7113 (1987); U.S. Pat. No. 5,496,552).However, BMPs have also been detected in or isolated from a variety ofother mammalian tissues and organs such as kidney, liver, lung, brain,muscle, teeth, and gut. Most BMPs (including BMP-2, BMP-4, BMP-6, BMP-7,BMP-9, BMP-12, BMP-13) also stimulate cartilage and bone formation asdemonstrated in a standard ectopic assay for bone formation (see, e.g.,Sampath and Reddi, Proc. Natl. Acad. Sci. USA, 80: 6591-6595 (1983)).Accordingly, such authentic BMPs are also referred to as “osteogenic”even though they may also promote soft tissue regeneration.

The protein referred to routinely as BMP-1 is not an authentic member ofthe BMP family of osteogenic, tissue regenerative proteins. BMP-1 wasoriginally isolated from highly purified BMP bovine bone extracts andwas originally reported to induce the formation of cartilage in vivo ina subcutaneous (ectopic) bone formation assay (Wozney et al., Science,242: 1528 (1988)). However, BMP-1 does not share significant amino acidsequence homology with other BMPs, nor does BMP-1 exhibit thecharacteristic signal peptide, prodomain, carboxy-terminal (maturedomain), or cysteine knot found in other BMPs. In fact, BMP-1 was shownto be identical to procollagen C-proteinase, an enzyme essential for theproper assembly of collagen within the extracellular matrix (ECM)(Kessler et al., Science, 271: 360-362 (1996)). The erroneous status ofBMP-1 within the TGF-β family resulted from flaws in the originalbioassay for osteogenesis (Wozney et al., op. cit.) in which thecartilage observed in the bioassay appears to have been old growth platecartilage contaminating the insoluble bone matrix that was misidentifiedas newly formed tissue (see, Reddi, Science, 271: 463 (1996)). As shownherein, unlike authentic osteogenic BMPs, the BMP-1-1 isoform does notinduce cartilage or bone formation in a standard ectopic bone formationassay.

The BMP-1 gene is related to the Drosophila gene tolloid (TLD), which isimplicated in the patterning controlled by the decapentaplegic (DPP)gene by virtue of its ability to activate TGF-β-like morphogens. TheBMP-1 protein is now known to be an essential control point ofmorphogenesis during the cascade of pattern formation (Ge and Greenspan,Birth Defect Res., 78: 47-68 (2006)).

BMP-1 is the prototype of a small subgroup of metalloproteinases foundin a broad range of species. In mammals, there are fourBMP-1/TLD-related (or BMP-1/TLD-like) metalloproteinases. The geneencoding BMP-1 also encodes a second, longer proteinase that is encodedby alternatively spliced mRNA. With a domain structure that isessentially identical to TLD, this proteinase was designated mammalianTolloid (mTLD) (Takahara et al., J. Biol. Chem., 269: 32572-32578(1994)). In addition, there are two genetically distinct mammalianBMP-1/TLD-related proteinases, designated mammalian Tolloid-like 1 and 2(mTLL1 and mTLL2). The prodomains of BMP-1/TLD-like proteinases must beproteolytically removed by subtilisin-like proprotein convertases (SPCs)(Leighton and Kadler, J. Biol. Chem., 278: 18478-18484 (2003)) toachieve full activity of these proteinases. The role of the prodomain ofBMP-1/TLD-like proteinases appears to be in maintaining theBMP-1/TLD-like proteinases in a latent form (Marques et al., Cell, 91:417-426 (1997); Sieron et al., Biochem., 39: 3231-3239 (2000); Leightonand Kadler, op. cit.).

BMP-1/TLD-related metalloproteinases are responsible for the proteolyticmaturation of a number of extracellular proteins related to formation ofthe extracellular matrix (ECM). These include various collagens, smallleucine-rich proteoglycans, SIBLING proteins, and the enzyme lysyloxidases, laminin-5, and an anti-angiogenic factor from the basementmembrane proteoglycan perlecan (Iozzo, Nat. Rev. Mol. Cell. Biol., 6:646-656 (2005); Greenspan, Top. Curr. Chem., 247: 149-183 (2005); Ge andGreenspan Birth Defect Res., op. cit.). BMP-1 is also involved inreleasing BMPs from extracellular matrix or in activating latent TGF-βfamily members, such as BMP-4, BMP-11 and GDF-8 (Wolfman et al., Proc.Natl. Acad. Sci. USA, 100: 15842-15846 (2003); Ge et al, Mol. Cell.Biol., 25: 5846-5858 (2005)).

The originally discovered form of BMP-1 is designated as BMP-1-1, andother BMP-1 isoforms encoded by splice variant RNA transcripts have beendescribed on the transcriptional level and designated with sequentialsuffixes: BMP-1-2, BMP-1-3, BMP-1-4, BMP-1-5, BMP-1-6, and BMP-1-7 (Liet al., Proc. Natl. Acad. Sci. USA, 93: 5127-5131 (1996); Wozney et al.,Science, 242: 1528 (1988); Janitz et al., J. Mol. Med., 76:141 (1998);Takahara et al J. Biol. Chem., 269: 32572 (1994); Hillman et al., GenomeBiol., 5: 16 (2004). As expected, the BMP-1 isoforms encoded by thesplice variant transcripts share a number of domains, including leaderpeptide, proregion, and protease (catalytic) region. Only the originalBMP-1, i.e., BMP-1-1, has previously been confirmed on the protein levelfollowing its isolation from bone. The sequences for BMP-1-2 and otherBMP-1 isoforms were deduced from nucleotide sequences of the splicevariant transcripts, but have not been described at the protein level.

Despite the correction in the literature of the identity of BMP-1-1,whether this protein or other BMP-1 isoforms have any role oftherapeutic relevance remains to be elucidated.

SUMMARY OF THE INVENTION

The present invention provides new methods of diagnosis and therapybased on discoveries relating to the circulation of BMP-1 isoforms inthe blood of individuals. The differential appearance of particularisoforms in the circulating blood of individuals has now been associatedwith particular bone defects or disorders of soft tissue. Accordingly,it is now possible for early diagnosis of particular disorders such asacute bone fracture, chronic renal failure, fibrodysplasia ossificansprogressive, osteogenesis imperfecta, acute pancreatitis, and livercirrhosis using a simple blood test to detect the presence of one ormore BMP-1 isoforms in a sample of blood. Moreover, the discoveriesdisclosed herein have led to the development of new treatment methodswhich enhance the effects of osteogenic bone morphogenic proteins (BMPs)in individuals suffering from particular bone defects. (See, Example 14,below.)

One embodiment of the present invention involves a method of diagnosinga defect or disorder in a bone or soft tissue of an individualcomprising determining the profile of BMP-1 isoforms in the blood of theindividual and comparing the profile to a standard blood profile ofBMP-1 isoforms associated with various defects and disorders. Such astandard blood profile based on pooled blood from healthy individualsand individuals undergoing treatment for various bone and soft tissuedisorders is presented in Table 1 (infra).

The diagnostic methods of the present invention are advantageouslycarried out using detector molecules capable of binding to one or moreBMP-1 isoforms. Suitable such detector molecules include antibodymolecules (including polyclonal antibodies and monoclonal antibodies,and binding fragments of antibodies such as Fab fragments, F(ab′)2fragments, and the like) and aptamers (i.e., nucleic acid molecules thathave a specific binding affinity for particular proteins).

Thus, in a particular embodiment for diagnostic methods of theinvention, a blood isoform profile for an individual is made, using oneor more detector molecules to assay a sample of blood from theindividual for the presence of one or more BMP-1 isoforms. CirculatingBMP-1 isoforms, or the complete absence of any circulating isoforms, isdemonstrated herein to be indicative of particular disorders. Theability to detect these defects or disorders from a blood sample isadvantageous because a positive diagnosis can be achieved much earlierin the course of the disorder. Acute pancreatitis, for example, may bedetected from the presence of circulating BMP-1-7 and may be diagnosedprior to the manifestation of more overt symptoms of the disease.Similarly, an acute bone fracture such as a hairline fracture or crackthat is not easily detectable (or not detectable without expensivex-rays) may be deduced in the first instance using a blood test andobserving the complete absence of BMP-1 isoforms. In particularembodiments, detector molecules such as antibody molecules or aptamersspecific for one or more BMP-1 isoforms are used in an assay to detectthe presence of one or more BMP-1 isoforms in a sample of blood, and thepresence of certain isoforms (or the complete absence of isoforms) isindicative of a disorder associated with such presence (or absence)herein.

Preferred detector molecules for the diagnostic methods of thisinvention are monoclonal antibody molecules. A suitable anti-BMP-1isoform antibody molecule for use herein may be an immunoglobulin, a Fabfragment, a F(ab′)2 molecule, a single chain antibody molecule (scFv), adouble scFv molecule, a single domain antibody molecule (dAb), a Fdmolecule, a diabody molecule, a fusion protein comprising any of saidantibody molecules, or combinations of one or more of the foregoing.

In a particular method according to the present invention, a method isprovided for diagnosing liver cirrhosis in an individual comprising:testing a blood sample from an individual to determine the presence inthe sample of the BMP-1 isoforms BMP-1-1, BMP-1-3, BMP-1-5, and BMP-1-7,wherein the absence of said BMP-1 isoforms in the sample is indicativeof liver cirrhosis in the individual.

Another particular embodiment of the present invention is a method fordiagnosing acute bone fracture in an individual comprising: testing ablood sample from an individual to determine the presence in the sampleof the BMP-1 isoforms BMP-1-1, BMP-1-3, BMP-1-5, and BMP-1-7, whereinthe absence of said BMP-1 isoforms in the sample is indicative of anacute bone fracture in the individual.

A further embodiment of the present invention is a method for diagnosingacute pancreatitis in an individual comprising: testing a blood samplefrom an individual to determine the presence in the sample of the BMP-1isoform BMP-1-7, wherein the presence of said BMP-1 isoform incirculating blood of said individual is indicative of acute pancreatitisin the individual.

A further embodiment of the present invention is a method for diagnosingchronic renal failure in an individual comprising: testing a bloodsample from an individual to determine the presence in the sample of theBMP-1 isoforms BMP-1-3 and BMP-1-5, wherein the presence of both saidBMP-1 isoforms in circulating blood of said individual is indicative ofchronic renal failure in said individual.

A particularly advantageous method disclosed herein is a method fordiagnosing fibrodysplasia ossificans progressive in an individualcomprising: testing a blood sample from an individual to determine thepresence in the sample of the BMP-1 isoform BMP-1-3, wherein elevatedlevels (for example at least 5 times) of said BMP-1 isoform incomparison with levels of the same isoform in a healthy individual isindicative of fibrodysplasia ossificans progressive in said individual.

Another particularly advantageous embodiment of the present invention isa method for diagnosing osteogenesis imperfecta in an individualcomprising: testing a blood sample from an individual to determine thepresence in the sample of the BMP-1 isoform BMP-1-3, wherein elevatedlevels (for example, at least 5 times) of said BMP-1 isoform incomparison with levels of the same isoform in a healthy individual isindicative of osteogenesis imperfecta in said individual.

A further embodiment of the present invention is a method of treating anindividual for a defect or disorder in bone or soft tissue of anindividual comprising:

-   -   (a) diagnosing a defect or disorder in a bone or soft tissue in        an individual by steps comprising:        -   (i) determining the profile of BMP-1 isoforms in the blood            and        -   (ii) comparing the profile to a standard blood profile of            BMP-1 isoforms associated with various defects and            disorders,    -   (b) administering to the individual an amount of at least one        BMP-1 isoform effective to enhance the therapeutic effect of an        osteogenic BMP toward the diagnosed defect or disorder, or        administering to the individual an amount of one or more        antibody molecules specific for one or more BMP-1 isoforms        effective to inhibit the effects of said one or more BMP-1        isoforms in the progression of the diagnosed defect or disorder.

The diagnosing step (a) of the foregoing method may be performed bycomparing the patient's blood BMP-1 isoform profile with, for example,the standard blood isoform association table shown in Table 1, below.The therapeutic step (b) of the foregoing method may be accomplished viasystemic or local administration of the therapeutic agent. In treatingbone defects in particular, local administration to the area of thedefect is preferred. Local administration of BMP-1 isoform BMP-1-1, forinstance, is shown herein to accelerate bone repair in in vivo fracturemodels. (See, Examples 12 and 14, below.) Local administration of aBMP-1 isoform and/or an authentic, osteogenic BMP such as BMP-7 mayadvantageously be effected using a whole blood coagulum as acarrier/matrix for localized delivery of those agents to the bonedefect. A whole blood-derived coagulum device is described herein whichprovides a mechanically stable (self-supporting) therapeutic with theconsistency of a gel, which in turn is easily applied to bone ends or ina gap between bone sections where rebridgement of bone is desired.

In particular embodiments of the foregoing diagnostic methods, thedetection step will be directed toward detecting one or more of BMP-1-1,BMP-1-3, BMP-1-5, and BMP-1-7, having the amino acid sequences shown inSEQ ID NO:1, SEQ ID NOS:2 or 4, SEQ ID NO:6, and SEQ ID NO:7,respectively, or detecting an epitope or a detectable fragment (such asa tryptic fragment) of said amino acid sequences.

In a particular embodiment, the present invention provides an osteogenicwhole blood-derived coagulum device (WBCD) for treating a bone defect inan individual prepared by mixing together in an aliquot of whole blood asubstance providing calcium ions (Ca⁺⁺), such as calcium chloride; atleast one BMP-1 isoform and optionally at least one osteogenic BMP; andoptionally a composition comprising fibrin and thrombin. The mixture isincubated until a coagulum having the consistency of a mechanicallystable gel forms, and thereafter the coagulum is easily applied as amatrix to the site where bone rebridgement or repair is desired. Suchmechanically stable gel will preferably be homogenous, cohesive,syringeable, injectable and malleable. The consistency of the coagulumensures that the mixture, entraining the therapeutic BMP (if present)and BMP-1 isoform, will remain in place adjacent the bone defect to berepaired.

The proportions of the ingredients of the coagulum may be varied, butthe amount of calcium ion substance should be such that theconcentration of calcium ion provides a coagulum gel having the desiredfeatures mentioned above. A preferred concentration of calcium ions inthe coagulum will fall in the range of 1-2.5 mM. Calcium chloride is apreferred exogenous Ca⁺⁺-supplying substance. When calcium chloride isused in a WBCD of the invention, the concentration is advantageously inthe range of 5-15 mM.

The amount of BMP-1 isoform in a coagulum according to the invention isadvantageously in the range of 1-500 μg/mL, preferably 2-200 μg/mL, morepreferably 5-20 μg/mL, although lesser or greater amounts may also beused: it is a basic discovery disclosed herein that the presence ofBMP-1 isoforms is helpful to catalyze the activity of authentic,osteogenic BMPs locally, e.g., in repairing bone defects and rebridgingbone fractures. Thus, any amount of a BMP-1 isoform effective to enhancethe osteogenic activity of BMP (whether activated from the extracellularmatrix or supplied exogenously, e.g., as a component of a wholeblood-derived coagulum device) may be used. Similarly, if one or moreBMP is used as a component of a coagulum device according to theinvention, the amount may advantageously be adjusted to fall in therange of 50-500 μg/mL, preferably 100-200 μg/mL. As with the BMP-1isoform component, however, lesser or greater amounts are contemplated,and any amount of a BMP effective to promote osteogenesis at theintended site of the bone defect may be used. Alternatively, the amountsof a BMP-1 isoform or a BMP used in a coagulum may be adjusted toprovide an overall dose of isoform or BMP based on the overall weight ofthe individual, considering the amount of coagulum to be used. Forexample, an amount of BMP-1 isoform to provide 2-200 μg/kg, preferably5-20 μg/kg, more preferably 8-12 μg/kg patient weight, may be used; andan amount of a BMP to provide, e.g., 1-1000 μg/kg, preferably 2-500μg/kg, more preferably 50-200 μg/kg, most preferably 100 μg/kg patientweight, may be used. In determining the amounts of ingredients for usein a WBCD, it will be understood that the amounts or volumes of theingredients cannot be so much (or so little) as to adversely affect thedesired features of the coagulum gel.

Accordingly, in a particular embodiment of the invention, an osteogenicwhole blood-derived coagulum device (WBCD) for treating a bone defect inan individual is prepared by the steps comprising:

-   -   (a) mixing together:        -   (i) whole blood,        -   (ii) 2-200 μg/mL of at least one BMP-1 isoform,        -   (iii) 5-15 millimoles/L calcium chloride,        -   (iv) optionally, a mixture comprising 5-10 mg/mL fibrin and            0.5-5 mg/mL thrombin; and    -   (b) incubating the mixture of step (a) until a mechanically        stable gel is formed.

If desired, an amount of a BMP, preferably in the range of 50-500 μg/mL,may be added to the mixture of (a) in the foregoing embodiment, to takeadvantage of the synergistic effect of the combination of BMP-1 isoformand BMP disclosed herein.

Many suitable substances for providing calcium ions are known. Calciumchloride is preferred.

Fibrin-thrombin mixtures useful in a WBCD described herein may be madeby simply mixing fibrin and thrombin in with the other ingredients ofthe WBCD. Alternatively, fibrin and thrombin may be premixed orpurchased as a mixture and the mixture then added to the otheringredients. Fibrin-thrombin mixtures useful in a WBCD include thoseknown in the art as “fibrin glue” or “fibrin sealant”. Commercialpreparations of fibrin-thrombin mixtures, fibrin glues, and fibrinsealants are readily available. Fibrin and thrombin used in preparing aWBCD as described herein are of pharmaceutically acceptable quality andare not a source of significant immunogenicity that would normallyelicit an immune response in most individuals.

An exogenously provided fibrin-thrombin mixture may enhance one or moreof the properties provided to the coagulum gel by calcium ion asmentioned above. In addition, a fibrin-thrombin mixture can also be usedto entrap the BMP-1 isoform (and optional BMP) component(s) of a WBCD.Such entrapment of such active ingredients enhances retention by theWBCD and thereby decreases the rate of migration of the activeingredients from the WBCD and the local defect site to which the WBCDhas been applied. Preferably, the exogenously provided fibrin-thrombinmixture used in a WBCD described herein provides fibrin in the range of5 mg/mL to 10 mg/mL, inclusive, and provides thrombin in the range of0.5 mg/mL to 5 mg/mL.

In preparing the osteogenic WBCD according to the invention, the wholeblood is most preferably autologous whole blood drawn from theindividual. Thus, it is contemplated that the WBCD will be prepared foruse in bone repair surgery, in the operating theater, immediately priorto use, and employing the patient's own whole blood to make the WBCD.This has the obvious advantage of avoiding the necessity of typing andcross-matching donor blood for administration to a particular patient.Nevertheless, it is recognized that in some situations, crossmatchedwhole blood may be used as, e.g., when a patient may already have lost asignificant amount of blood or may already be receiving a bloodtransfusion. In such situations, the use of crossmatched whole blood ina WBCD introduces the same or similar risks of serum sickness associatedwith any transfusion employing crossmatched whole blood.

In a particular embodiment, the osteogenic WBCD according to theinvention may be prepared by first combining any fibrin/thrombincomposition, the calcium ion substance, and the BMP-1 isoform andoptionally BMP to form a first mixture, then adding whole blood to thefirst mixture to form a second mixture, and incubating the secondmixture until a mechanically stable (self-supporting) gel is formed.

In another embodiment, all the components necessary for preparation of aWBCD except the whole blood component may be conveniently andadvantageously collected in a kit. The kit may be opened and used in theoperating room at the moment it is needed, to form a WBCD usingautologous blood obtained from the patient. Such a kit could include,for example, the following items:

-   -   (a) a vial containing one or more lyophilized BMP-1 isoform,    -   (b) a buffer for reconstituting the lyophilized BMP-1        isoforms(s),    -   (c) a syringe for reconstituting the lyophilized BMP-1        isoform(s) in the buffer,    -   (d) a vaccutainer for collecting a patient's blood,    -   (e) a sterile solution of 1 M calcium chloride,    -   (f) a fibrin-thrombin mixture,    -   (g) a container for mixing whole blood with the reconstituted        BMP-1 isoform(s) and other ingredients,    -   (h) a spatula or syringe (or both) suitable for applying an        osteogenic coagulum to bone ends during open bone repair        surgery, and    -   (i) instructions for the preparation and use of a WBCD comprised        of whole blood mixed with one or more BMP-1 isoforms, calcium        chloride and, optionally, a mixture comprising fibrin and        thrombin, to form a mechanically stable gel suitable for        application to a bone defect.

The discoveries disclosed herein provide new approaches to therapy ofbone defects and soft tissue disorders, based on the discovered role ofBMP-1 isoforms and their presence in circulating blood.

In a particular embodiment, a method is provided for treating ischemicacute renal failure in an individual comprising administering a BMP-1isoform systemically to the individual after diagnosis of renal injury.(See, Example 8, below.) In a related embodiment, a method is providedfor treating chronic renal failure in an individual comprisingadministering systemically to the individual one or more antibodymolecules specific for one or more BMP-1 isoforms. (See, Example 9,below.) In a particular embodiment of this method, the antibody moleculeis an antibody molecule specific for the BMP-1-1 isoform, an antibodymolecule specific for the BMP-1-3 isoform, or a combination of suchantibody molecules.

A further embodiment of the invention provides a method of treatingischemia/reperfusion damage to a kidney in an individual comprising:administering to the individual one or more antibody molecules specificfor one or more BMP-1 isoforms in an amount effective to inhibitischemia/reperfusion injury in said individual. In particularembodiments, one or more antibody molecules recognizing one or moreBMP-1 isoforms is administered systemically to the individual prior toan ischemia/reperfusion event. In particular, an antibody moleculebinding to BMP-1-1, an antibody molecule binding to BMP-1-3, or acombination of such antibody molecules may be administered to theindividual.

The present invention also provides a method of pretreating anindividual to resolve clots that may occur during thoracic or abdominalsurgery comprising administering a BMP-1 isoform to the individual priorto surgery in an amount effective to resolve clots that occur.

A further embodiment of the present invention provides a method oftreating acute pancreatitis in an individual comprising administering tothe individual a therapeutically effective amount of at least oneantibody molecule specific for a BMP-1 isoform. In particular, in thisembodiment, an anti-BMP-1-7 antibody molecule may be used.

A further embodiment of the present invention provides a method oftreating pancreatitis in an individual comprising administering to anindividual suffering from pancreatitis, after the acute phase of theinflammatory process, an amount of a BMP-1 isoform in an amounteffective to promote pancreatic regeneration. In particular, in thisembodiment, the BMP-1-7 isoform may be administered.

In the course of our investigation of circulating BMP-1 isoforms, wealso isolated, from a placental cDNA library, a polynucleotide encodinga previously unreported variant of BMP-1 isoform BMP-1-3. The codingsequence for this isoform is shown in SEQ ID NO:5; the amino acidsequence for this variant isoform is shown in SEQ ID NO:4. The BMP-1-3isoform expressed from the isolated placental cDNA exhibits someadditional properties as compared to the previously reported BMP-1-3isoform (SEQ ID NO:2). (See, Example 5, below.) Accordingly, anadditional aspect of the present invention is to provide an isolatedpolynucleotide encoding the polypeptide having the amino acid sequenceof SEQ ID NO:4. One such polynucleotide has the sequence of SEQ ID NO:5.

In its broadest aspects, the present invention relates to the use of adetector molecule that specifically binds a BMP-1 isoform in an in vitrodiagnostic method to test for the presence of one or more BMP-1 isoformsin circulating blood of an individual, for diagnosing a defect ordisorder in bone or soft tissue in said individual. In preferredembodiments such a detector molecule is an antibody molecule or anaptamer. Advantageously, such detector molecules are detectably labeled.

The present invention, in its therapeutic aspects, provides for the useof a BMP-1 isoform in the manufacture of a medicament for the treatmentof bone defects. Also, the present invention provides for the use of anantibody molecule that binds a BMP-1 isoform in the manufacture of amedicament for treatment of soft tissue disorders as herein described.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph of the concentration (mg/dL) of creatinine versustime (days) in blood of rats subjected to ischemic acute renal failure.Diagonal line bars show levels of creatinine in the blood of rats of thecontrol group (ischemia, no treatment) at indicated times after theischemic event. Stippled bars show levels of creatinine in blood of ratstreated systemically with antibodies to BMP-1-1 and to BMP-1-3 prior toischemia and for five days thereafter. Asterisks indicate significant(P<0.01) difference between creatinine levels between animals treatedwith antibodies and those of the untreated control group. The resultsindicate that systemic administration of BMP-1-1 and BMP-1-3neutralizing antibodies prevented loss of kidney function in rats withischemia/reperfusion acute renal failure if administered prior toinjury. See Example 7, below, for details.

FIGS. 2A and 2B show histological sections of kidney tissues from ratssubjected to ischemia/reperfusion acute renal failure as described forFIG. 1, above, and in Example 7, below. FIG. 2A shows a representativehistological section of kidney tissue from a rat of the control groupthat was subjected to acute ischemia/reperfusion injury without antibodytherapy (physiological saline vehicle, pH 7.2, only). Significant lossof structural integrity of kidney tissue is evident in FIG. 2A. FIG. 2Bshows a representative histological section of kidney tissue from a ratof the prophylactic therapy group that was systemically administeredantibodies to BMP-1-1 and BMP-1-3 prior to acute ischemia/reperfusioninjury and for five days thereafter. Tissue in FIG. 2B indicatessignificant preservation of kidney structures, as compared to theuntreated tissues depicted in FIG. 2A. See, Example 7, below, fordetails.

FIG. 3 shows a graph of the percent survival of rats over time (days)after ischemic acute renal failure injury as described in Example 8,below. Diamonds (♦, “control”) show survival of rats in the negativecontrol group that did not receive therapy after ischemia/reperfusioninjury. Squares (▪, “BMP-7”) show survival of rats in the positivecontrol group that received BMP-7, a known therapeutic agent fortreatment of ischemia/reperfusion injury in kidney. Triangles (▴,“BMP-1”) show survival of rats that received BMP-1-1 after injury.Diagonal crosses (x, “BMP-1 Ab”) show survival of rats that receivedantibody to BMP-1-1 after injury. The results indicate thatadministration of BMP-1-1 isoform after injury increased the survivalrate of rats with ischemia/reperfusion acute renal failure. See, Example8, for details.

FIGS. 4A-4F show fractures in femurs after 8 weeks from rats treatedsystemically with BMP-1-1 (bones in FIGS. 4A and 4D), BMP-7 (bones inFIGS. 4B, 4C, and 4E), and antibody to BMP-1-1 (bone in FIG. 4F).Systemic administration of BMP-1-1 to rats resulted in acceleratedhealing of fractures as compared to systemic administration of BMP-7 torats. Systemic administration of BMP-1-1 neutralizing antibody delayedthe fracture healing due inhibition of BMP-1-1 activity at the fracturesite.

FIGS. 5A and 5B show ulnar defect in representative bone after 6 weeksfrom rabbits of a control group treated locally with a wholeblood-derived coagulum device (WBCD) only, without BMP-1 isoform orBMP-7, as described in Example 14, below.

FIGS. 6A and 6B show ulnar defect in representative bone after 6 weeksfrom rabbits treated locally with a WBCD containing BMP-1-1 as describedin Example 14, below.

FIGS. 7A and 7B show ulnar defect in representative bone after 6 weeksfrom rabbits treated locally with a WBCD containing BMP-7 as describedin Example 14, below.

FIGS. 8A and 8B show ulnar defect in representative bone after 6 weeksfrom rabbits treated locally with a WBCD containing BMP-1-1 and BMP-7 asdescribed in Example 14, below.

DETAILED DESCRIPTION OF THE INVENTION

In order that the invention may be fully understood the following termsare defined.

“Antibody” or “antibody molecule”, as used and understood herein, refersto a specific binding member that is a protein molecule or portionthereof or any other molecule, whether produced naturally,synthetically, or semi-synthetically, which possesses an antigenicbinding domain formed by an immunoglobulin variable light chain regionor domain (V_(L)) or portion thereof, an immunoglobulin variable heavychain region or domain (V_(H)) or portion thereof, or a combinationthereof. The term “antibody” also covers any polypeptide or proteinmolecule that has an antigen-binding domain that is identical, orhomologous to, an antigen-binding domain of an immunoglobulin.Antibodies may be “polyclonal”, i.e., a population of antigen-bindingmolecules that bind to different sites on the antigen, or “monoclonal”,i.e., a population of identical antigen-binding molecules that bind toonly one site on an antigen. Examples of an antibody molecule, as usedand understood herein, include any of the well known classes ofimmunoglobulins (e.g., IgG, IgM, IgA, IgE, IgD) and their isotypes;fragments of immunoglobulins that comprise an antigen binding domain,such as Fab or F(ab′)2 molecules; single chain antibody (scFv)molecules; double scFv molecules; single domain antibody (dAb)molecules; Fd molecules; diabody molecules; and fusion proteinscomprising such molecules. Diabodies are formed by association of twodiabody monomers, which form a dimer that contains two complete antigenbinding domains wherein each binding domain is itself formed by theintermolecular association of a region from each of the two monomers(see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448(1993)). Use of such antibody molecules offers the vast array ofantibody detection systems and formats available in the art that may beadapted to selectively detect particular BMP-1 isoforms in mixtures,including whole blood, plasma, serum, and various tissue extracts.Examples of formats for using antibody molecules to detect BMP-1isoforms may include, but are not limited to, immunoblotting (e.g.,Western blots, dot blots), immunoprecipitations, affinity methods,immunochips, and the like. Any of variety methods known in the art maybe employed to produce antibody molecules to a specific BMP-1 isoform ora portion thereof comprising at least one epitope (antibody bindingsite) of the BMP-1 isoform.

“Circulate” and “circulating” describe anything that travels or isotherwise transported through the vascular system of an individual.

The terms “disorder” and “disease” are synonymous and refer to anypathological condition, irrespective of cause or etiological agent. A“defect” in a tissue refers to a site of abnormal or deficient tissuegrowth. A “disease” or “disorder” may be characterized by one or more“defects” in one or more tissues.

As used herein, the terms “treatment” and “treating” refer to anyregimen that alleviates one or more symptoms or manifestations of adisease or disorder, that inhibits progression of a disease or disorder,that arrests progression or reverses progression (causes regression) ofa disease or disorder, or that prevents onset of a disease or disorder.Treatment includes prophylaxis and includes but does not require cure ofa disease or disorder.

A “therapeutically effective amount” is an amount of a compound (e.g., aBMP-1 isoform or a BMP-1 isoform binding molecule when usedtherapeutically) which inhibits, totally or partially, the progressionof the condition, which alleviates, at least partially, one or moresymptoms of the disorder, or which enhances or catalyzes the therapeuticor otherwise beneficial effects of another compound (e.g., an osteogenicBMP). A therapeutically effective amount can also be an amount which isprophylactically effective. The amount which is therapeuticallyeffective will depend upon the patient's size and gender, the conditionto be treated, the severity of the condition and the result sought. Fora given patient, a therapeutically effective amount can be determined bymethods known to those of skill in the art.

The term “isolated” when used to describe the various polypeptidesdisclosed herein, means a polypeptide that has been identified andseparated and/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould typically interfere with diagnostic or therapeutic uses for thepolypeptide, and may include enzymes, hormones, and other proteinaceousor non-proteinaceous solutes. Isolated polypeptide includes polypeptidein situ within recombinant cells engineered to express it, since atleast one component of the polypeptide's natural environment will not bepresent. Ordinarily, however, isolated polypeptide will be prepared byat least one purification step. An “isolated polynucleotide” or isolatedpolypeptide-encoding nucleic acid is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofsuch nucleic acid, e.g., the human genome. An isolated polynucleotide isother than in the form or setting in which it is found in nature.Isolated polynucleotides therefore are distinguished from the specificpolypeptide-encoding nucleic acid molecule as it exists in naturalcells. However, an isolated polynucleotide includes polypeptide-encodingnucleic acid molecules contained in cells that ordinarily express thepolypeptide but where, for example, the nucleic acid molecule is in achromosomal location different from that of natural cells.

“Gel” means a semi-solid jelly-like material.

“Homogenous”, as applied to a coagulum gel, means that the coagulum gelhas a uniform consistency as opposed to a nonuniform fibrous networkconnecting clumps of clots.

“Syringeable” as used herein to describe a coagulum gel means that thecoagulum gel can be drawn up into a syringe with a needle in the rangeof 18 to 23 gauge, inclusive, without clogging the needle or breaking upinto clumps.

“Injectable” as used herein to describe a coagulum gel means that thecoagulum gel can be expelled from a syringe through the aperture of thesyringe or through a needle in the range of 18 to 23 gauge, inclusive,without clogging the aperture or needle and without breaking up intoclumps.

“Malleable” as used herein to describe a coagulum gel means that thecoagulum gel is capable of being shaped or formed to fill or cover abone defect. A malleable coagulum gel is self-supporting (ormechanically stable) and will substantially retain the shape into whichit was formed.

A composition or method described herein as “comprising” one or morenamed elements or steps is open-ended, meaning that the named elementsor steps are essential, but other elements or steps may be added withinthe scope of the composition or method. To avoid prolixity, it is alsounderstood that any composition or method described herein as“comprising” (or “which comprises”) one or more named elements or stepsalso describes the corresponding, more limited, composition or method“consisting essentially of” (or “which consists essentially of”) thesame named elements or steps, meaning that the composition or methodincludes the named essential elements or steps and may also includeadditional elements or steps that do not materially affect the basic andnovel characteristic(s) of the composition or method. It is alsounderstood that any composition or method described herein as“comprising” or “consisting essentially of” one or more named elementsor steps also describes the corresponding, more limited, and close-endedcomposition or method “consisting of” (or “which consists of”) the namedelements or steps to the exclusion of any other unnamed element or step.In any composition or method disclosed herein, known or disclosedequivalents of any named essential element or step may be substitutedfor that element or step.

Unless indicated otherwise, the meaning of other terms is the same asunderstood and used by persons in the art, including the fields ofmedicine, biochemistry, molecular biology, and tissue regeneration.

The invention is based on the discovery that BMP-1 isoforms in the bloodof an adult individual (human or other mammal) are useful as biologicalmarkers (biomarkers) for the state or condition of the tissues of theindividual. In particular, the presence or absence of one or moreisoforms of BMP-1 in the blood, i.e., the BMP-1 isoform blood profile,of an adult individual is indicative of the health or a particularpathological state of bone and various soft tissues of the individual.BMP-1-1, which is identical to the metalloproteinase procollagenC-proteinase (also referred to as BMP-1 procollagen C-proteinase) wasoriginally discovered in the bone matrix. However, the BMP-1-1 isoformis not found circulating in the blood of the healthy adult individual,nor in patients with various diseases. Previously, the existence ofisoforms other than BMP-1-1 was inferred only at the level of tissue RNAtranscripts.

Table 1, below, provides profiles of circulating BMP-1 isoformsassociated with normal health and with several disorders, i.e., an acutebone fracture, chronic renal failure, fibrodysplasia ossificansprogressive (FOP), osteogenesis imperfecta (JO), acute pancreatitis, andcirrhosis of the liver. A description of the study that generated thediagnostic profiles in Table 1 is provided in Example 6 (below).

TABLE 1 BMP-1 isoforms in various tissue defects and disorders BMPIsoform Pathology of Patient BMP-1-1 BMP-1-3 BMP-1-5 BMP-1-7 healthy(normal) − + − − acute bone fracture − − − − chronic renal failure − + +− FOP − ++ − − OI − ++ − − acute pancreatitis − − − + liver cirrhosis −− − − FOP = fibrodysplasia ossificans progressive; IO = osteogenesisimperfecta ++ indicates much higher than normal levels (i.e., at least5-fold higher than in healthy individuals)

Blood obtained from an individual can be easily analyzed for thepresence of various BMP-1 isoforms, e.g., using isoform-specificantibodies or other isoform detector molecules. The profile of BMP-1isoforms in the blood sample can then be compared to the profiles inTable 1 to diagnose any of the indicated pathological states.

Table 1 shows that circulating BMP-1 isoforms are useful as biologicalmarkers (i.e., biomarkers) of a broad spectrum of diseases. The use ofthe BMP-1 isoform blood profiles to diagnose the pathologies in Table 1is not dependent on an understanding of the mechanism by which suchprofiles are generated. Nevertheless, there are implications to the datapresented herein beyond providing a convenient method of diagnosingvarious disorders. In particular, data presented herein demonstrate forthe first time the existence of circulating enzymes that are variantproducts of a single gene, BMP-1. Moreover, without wishing to be boundby any particular mechanism or theory of operation, the data in Table 1dispel a long-held model for the action of authentic osteogenic BMPs inwhich each tissue or organ was assumed to release a particular authenticBMP (e.g., BMP-4, BMP-5, BMP-6) into the circulation during injury andin the process of regeneration of that tissue or organ. On the contrary,as shown in Table 1, in healthy individuals only the BMP-1-3 isoformcirculates, and no authentic osteogenic BMPs have been found in theblood of healthy individuals (see, Example 1, below). Moreover, as shownherein, as much as 80% of intravenously administered BMP-1-3 becomeslocalized at the orthotopic site of fractured femurs in rats and resultsin an accelerated rate of bone healing compared to untreated controlanimals (see, Example 7, below). In addition, in cultures of ratcalvariae, which are rich in ECM, exogenously provided BMP-1-3 promotesrelease into the culture medium of authentic osteogenic BMP-4 and BMP-7(see, Example 9, below). These data are more consistent with the tissuerepair model shown herein, that circulating BMP-1 isoforms can actcatalytically as key processing enzymes of the ECM (which is arepository of authentic osteogenic BMP molecules (see, e.g., Martinovicet al., Arch. Cytol. Histol., 1: 23-36 (2006))) to effect a localrelease of one or more authentic osteogenic BMPs. In bone repair, forexample, BMP-1 isoform-catalyzed release of authentic BMP locally actsin turn locally to promote bone regeneration and repair during theformation of callus during the rebridgement of fractured bone ends.

As shown in Table 1, a number of pathological conditions arecharacterized by a disappearance of the BMP-1-3 isoform from the blood,i.e., acute bone fracture, acute pancreatitis, and liver cirrhosis. Ifin addition to BMP-1-3, the BMP-1-5 isoform is also present in the bloodof an individual, then the isoform profile is diagnostic of chronicrenal failure (CRF). If BMP-1-3 is found in the blood at much higherconcentrations than in a normal individual (i.e., at least 5 times thenormal level), then the isoform profile is diagnostic of FOP or OI. IfBMP-1-7 is the only isoform present, then the profile is diagnostic ofacute pancreatitis.

With respect to soft tissue organs, an absence of BMP-1-3 in the bloodmay indicate a condition in which the BMP-1-3 accumulates in aparenchymal organ to facilitate processing of the extracellular matrix(ECM), which in turn stimulates fibrosis. A common feature of the softtissue pathologies in Table 1 is a progressing fibrosis of the tissue,which untreated can lead to organ failure. Such fibrosis ischaracteristic of cirrhosis of the liver and acute pancreatitis.Accordingly, when a blood profile indicates the absence of BMP-1-3, andthere is no evidence of bone fracture, chronic renal failure, FOP, orOI, then Table 1 directs the diagnosis to the specified pathologies ofparenchyma organs, such as liver or pancreas. In such situations, thehealthcare professional is alerted to perform additional tests forpathology in such organs. Accordingly, such additional tests may includedetermining whether one or more parenchyma organs exhibits increasedfibrosis as evidenced by performing standard tests for an accumulationof collagen, laminin, fibronectin, and other extracellular moleculesleading to increased fibrosis.

For Table 1, the sera from patients with acute pancreatitis werecollected at an early stage of the disease, i.e., prior to robust serumelevation of the pancreatic enzymes such as pancreatic amylase andlipase. Surprisingly, the blood of these patients contained the BMP-1-7isoform, which has not been previously detected at the protein level(that is, as an expressed protein rather than a theoretical BMP variantdeduced from detection of mRNA transcripts). The appearance in the bloodof BMP-1-7 is useful as an early diagnostic marker for acute injury ofthe pancreas.

The BMP-1-3 and BMP-1-5 isoforms were found in patients with chronickidney failure on dialysis and suggest a specific function of theseisoforms in the disorder, e.g., involvement in the fibrotic processes inbone called renal osteodystrophy. The BMP-1-5 isoform has also beendetected in the circulation of rats with chronic renal failurereflecting the severity of the disease. Our detection of BMP-1-5 in theblood of patients is also the first demonstration of the BMP-1-5 isoformon the protein level.

According to the profiles in Table 1, a BMP-1 isoform profile thatindicates there are no BMP-1 isoforms circulating in the blood of apatient is evidence that the individual has an acute bone fractureand/or has liver cirrhosis. Both of these conditions involve fibrosis.Such fibrosis may be beneficial as part of callus formation in thehealing of an acute bone fraction, whereas in soft tissue, fibrosis isdestructive and is characteristic of liver cirrhosis.

Determining the circulating BMP-1 isoform profile may be used not onlywhen an individual presents symptoms of a tissue defect or disease, butalso as part of an individual's routine blood test conducted by anattending healthcare professional, e.g., as part of an annual physicalexamination. BMP-1 isoforms are readily detected in samples of bloodobtained from an individual using any of a variety of methods andcompositions known in the art. Such methods include, but are not limitedto, high performance liquid chromatography (HPLC), mass spectrometry(MS) of tryptic peptides of BMP-1 isoforms, and affinity methods,particularly those that employ affinity molecules that specifically binda particular BMP-1 isoform to the exclusion of other isoforms. Suchaffinity molecules include, but are not limited to, antibody moleculesand aptamers. Antibody molecules specific for each BMP-1 isoform areparticularly preferred as there is a wide variety of assay formatsavailable in the art that can employ an antibody molecule to detect orisolate a target protein present in the blood of an individual. Suchformats include, but are not limited to, filter paper (e.g.,nitrocellulose, cellulose acetate), microtiter plates, polymericparticles (e.g., agarose, polyacrylamide), silicon chips, etc. It isunderstood that for any particular method used to detect or isolate aBMP-1 isoform from the blood of an individual, it may be preferred tomake such detection or isolation from the plasma or serum portion ofwhole blood.

Recombinant BMP-1 isoforms described herein were cloned and expressed ineukaryotic and prokaryotic host cells. Such recombinant cells may beemployed to produce sufficient amounts of the isoforms for use in themethods described herein. The specific coding sequences for each of theBMP-1 isoforms discussed herein are known, and the encoded amino acidsequences have been deduced. See, e.g., EMBL Nucleotide SequenceDatabase (worldwide web.ebi.ac.uk/embl). For convenience, the amino acidsequence for BMP-1-1 is included herein as SEQ ID NO:1. The amino acidsequence for BPM-1 isoform BMP-1-3 is shown in SEQ ID NO:2, and a cDNAsequence coding for BMP-1-3 is shown in SEQ ID NO:3. The amino acidsequence for BMP-1 isoform BMP-1-5 is shown in SEQ ID NO:6. The aminoacid sequence for BMP-1 isoform BMP-1-7 is shown in SEQ ID NO:7. A newvariant form of BMP-1-3 derived from human placenta and havingproperties that differ from the previously known form of BMP-1-3 hasbeen discovered, having the amino acid sequence of SEQ ID NO:4 and acoding sequence shown in SEQ ID NO:5.

BMP-1 isoforms and peptides thereof may be produced by standardrecombinant, synthetic, or semi-synthetic methods available in the art.BMP-1 isoforms and peptides thereof may also be used to produce variousaffinity molecules, including polyclonal and monoclonal antibodymolecules, using standard methods available in the art.

All or a portion of a nucleotide sequence encoding the isoforms of SEQID NOS:1, 2, 4, 6, and 7 may be incorporated into the nucleotidesequence of any of a variety of nucleic acid molecules, such as vectors,primers, nucleic acid probes for hybridization, and the like. Suchrecombinant nucleic acid molecules may be used to clone nucleic acidmolecules encoding a BMP-1 isoform of interest, to identify or detectBMP-1 isoform nucleotide sequences (e.g., by various hybridizationmethods), and/or to amplify a nucleic acid molecule encoding a BMP-1isoform of interest (e.g., using a polymerase chain reaction (PCR)protocol). Nucleic acid molecules may be synthesized chemically (e.g.,using an automated nucleic acid synthesizer), produced by PCR, and/orproduced by various recombinant nucleic acid methods known in the art.Nucleic acid molecules may be synthesized with various modificationsknown in the art to provide molecules that resist cleavage by variousnucleases and chemicals, such as replacing phosphodiester linkages withthiol linkages. Methods of detecting a specific nucleotide sequence(DNA, cDNA, or RNA) encoding all or a portion of a BMP-1 isoform arewell known in the art and include, without limitation, Southern blots(for DNA and cDNA), Northern blots (for RNA), polymerase chain reaction(PCR) methods, dot blots, colony blots, and in vitro transcription ofDNA or cDNA molecules. Nucleic acid molecules as described herein mayalso be immobilized by standard methods to any of a variety surfacesincluding but not limited to a cellulose-containing paper (e.g.,nitrocellulose, cellulose acetate), nylon, a well of a plasticmicrotiter dish, polymeric particles (e.g., agarose particle, acrylamideparticles), and a silicon chip.

The profiles in Table 1 also suggest possible targets for drug discoveryand new methods of treating defects and disorders. For example, as notedabove, BMP-1 isoforms are implicated as key enzymes to promote fibrosis.Accordingly, fibrotic diseases may be treated by inhibiting orinactivating one or more BMP-1 isoforms that are implicated in tissuefibrosis. A preferred method of treating a fibrotic disease comprisesadministering to a patient an antibody to a BMP-1 isoform associatedwith tissue fibrosis. Such fibrotic diseases include, withoutlimitation, fibrotic kidney disease, liver cirrhosis, acutepancreatitis, and FOP. For example, in a method of treating a patientwith chronic renal failure and on dialysis therapy, an antibody to aBMP-1 isoform(s) may be administered to the patient to delay the kidneyfailure and prevent the development of renal osteodystrophy, which leadsto fragile bones and fibrotic bone marrow that inhibits the regenerativeprocess. In patients with FOP, an antibody molecule may be administeredto inhibit a BMP-1 isoform to prevent or inhibit ectopic ossifications,which depend on the fibrotic process to develop the characteristic“second skeleton” of FOP patients. Preferably, an antibody moleculeuseful in methods described herein is an antibody molecule that has verylow or, most preferably, no immunogenicity, so that the antibodymolecule may be administered in multiple doses to a patient withoutinvoking an immune response in the patient that would inactivate theantibody molecule. It is also understood that administration of atherapeutic agent, such as an antibody, to inhibit or inactivate a BMP-1isoform, may also inhibit healing of bone fractures, which depends onfibrosis in the formation of a bone callus in normal healing offractures. Accordingly, it will be appreciated by the healthcareprofessional that a therapy described herein to inhibit a BMP-1isoform(s) is not recommended until any bone fractures that may bepresent in a patient have healed or unless the healing of any fracturesin the patient is outweighed by a more critical need for therapy toinhibit or inactivate a BMP-1 isoform(s).

Another method of treatment of the invention comprises administering arecombinant BMP-1 isoform to a patient lacking a particular BMP-1isoform that could accelerate tissue repair or that could prevent adisease. As shown herein, BMP-1-3 disappears from circulation andbecomes localized in the orthotopic site of acute bone fracture.

Administration of recombinant BMP-1-1 to an individual that hassustained an acute form of a disease can accelerate bone repair whetherthe BMP-1 isoform is administered systemically (see, Example 7, below)or locally (see, Example 8, below). Administration of a BMP-1 isoformmay also be employed therapeutically to resolve blood clots that canoccur in patients following an ischemic acute renal failure during majoropen surgery, such as thoracic or abdominal surgery. In such cases, aBMP-1 isoform is preferably administered prior to surgery as apreventative therapy for resolving clots that might form during thesurgery.

In patients with acute pancreatitis, inhibition of the BMP-1-7 isoformmay be used prophylactically to prevent or to inhibit progression of thedisease, while systemic administration of BMP-1-7 following the acutephase of the inflammatory process may be used to promote pancreaticregeneration. The dual function of BMP-1 isoforms was shown in acuterenal failure in rats, where BMP-1-1 and BMP-1-3 antibodies injectedprior to kidney ischemia preserved the kidney function, while systemicadministration of BMP-1-1 isoform following the ischemia resulted in asignificantly greater survival of rats (see, Example 11, below). Thus, adual function of BMP-1-1 isoform in an acute ischemic disease suggeststwo treatment methods, i.e., a preventative (prophylactic) treatment anda therapeutic (regenerative) treatment. Accordingly, a method ofpreventing acute kidney ischemic disease may comprise administering(e.g., parenterally) to an individual an antibody to one or more BMP-1isoforms, e.g., antibody to circulating BMP-1-3 isoform and an antibodyto circulating BMP-1-1 isoform, to prevent fibrosis or to preventsubstantial progression of fibrosis. In contrast, a method of treatingacute ischemic kidney disease may comprise administering (e.g.,parenterally) to an individual one or more recombinant BMP-1 isoforms tosupport better regeneration of the kidney(s) in a subacute stage of thedisease. A method of treating chronic renal failure may compriseadministering (e.g., parenterally) to an individual an antibody to oneor more BMP-1 isoforms (e.g., antibody molecules to BMP-1-1 and toBMP-1-3) to inhibit fibrosis and progression of the disease. Ahealthcare professional is able to assess the condition of anindividual's kidneys to determine whether the individual is at risk ofacute ischemia and, therefore, is a candidate for preventative treatment(e.g., antibody molecules to inhibit BMP-1-3 and BMP-1-1 isoforms), orwhether the individual already suffers from significant acute ischemickidney disease, so as to be a candidate for the therapeutic(regenerative) treatment (administration of BMP-1 isoform(s)).

An important aspect of the findings described herein (see, Examples,below) is that contrary to the teachings and assumptions of the priorart, an osteogenic BMP of the BMP family (e.g., BMP-2, BMP-4, BMP-6,BMP-7, and the like) should not be administered systemically to providetherapeutic treatment for local repair of bone fractures or disorderssince any compromise in the wall of a blood vessel may release theosteogenic BMP locally thereby potentially inducing ossification oflocal soft tissue. Such compromise of blood vessels readily occurs atinjection sites, bruises, and wounds where the combination of locallyavailable stem cells and an osteogenic BMP can result in undesiredossification of soft tissue (e.g., muscle tissue). In contrast, BMP-1isoforms such as BMP-1-3 or BMP-1-1 may be administered systemically torelease an osteogenic BMP from extracellular matrix at a local site ofbone fracture. BMP-1-1 and its isoforms are not authentic BMPs but areenzymes.

A BMP-1 isoform may be employed as an active ingredient in a wholeblood-derived coagulum device (WBCD) to treat a bone defect, such as afracture or a bone that is characterized by inadequate bone growth(e.g., as occurs in various metabolic bone disorders), in an individual.Such WBCDs comprising one or more BMP-1 isoforms (e.g., BMP-1-1,BMP-1-3) may be implanted or injected into a site of fracture or otherdefect characterized by inadequate bone growth to promote boneregeneration. WBCDs prepared for the delivery of one or more BMPs aredescribed in detail in commonly assigned, copending internationalapplication no. PCT/US07/016,601, filed 23 Jul. 2007 (PCT PublicationNo. WO 2008/011192). The disclosure of that application is herebyincorporated by reference. The discovery as part of this invention thatBMP-1 isoforms catalyze authentic, osteogenic BMPs from EMC (orintroduced from exogenous sources) to enhance bone repair activityprovides a basis for describing herein improved WBCDs which include atleast one BMP-1 isoform or a combination of at least one BMP-1 isoformwith at least one osteogenic BMP.

Thus, in a preferred embodiment, this invention provides an osteogenicWBCD for treating a bone fracture or other bone defect that ischaracterized by inadequate bone growth in an individual comprising:

(a) whole blood;

(b) a BMP-1 isoform in the amount of 1-500 μg/mL, preferably 2-200μg/mL, more preferably 5-20 μg/mL, and optionally an authentic BMP inthe amount of 50-500 μg/mL;

(c) an exogenous substance to supply calcium ions (Ca⁺⁺) at aconcentration of 1-2.5 mM; and

(d) optionally, a mixture of 5-10 mg/mL fibrin and 0.5-5 mg/mL thrombin.

A whole blood-derived coagulum device described herein is preferablyprepared by the steps comprising:

-   -   (a) mixing together:        -   (1) whole blood,        -   (2) 1-500 μg/mL, preferably 2-200 μg/mL, more preferably            5-20 μg/mL, of at least one BMP-1 isoform,        -   (3) 5-15 millimoles/L calcium chloride, and        -   (4) optionally, a mixture of 5-10 mg/mL fibrin and 0.5-5            mg/mL thrombin;    -   (b) incubating the mixture of step (a) until a mechanically        stable (i.e., a non-fluid, self-supporting, adherent) coagulum        gel is formed.

In the foregoing embodiment, one or more authentic, osteogenic BMPs,preferably in an amount of 50-500 μg/mL, may also be added to the mixingstep (a).

In a preferred embodiment, the coagulum device is prepared by firstcombining the fibrin-thrombin mixture, calcium ion, and BMP-1 isoform orBMP components to form a first mixture; followed by combining said firstmixture with whole blood until the concentrations of the ingredientsfall within the ranges set forth above and a mechanically stablecoagulum of gel consistency is formed.

Preferably, the whole blood used in the preparation of a WBCD describedherein is the autologous whole blood drawn from the individual who is toreceive the WBCD, as autologous whole blood will not be immunogenic,that is, will not be rejected as non-self tissue by the immune system ofthe recipient. Nevertheless, it is recognized that in some situations,crossmatched whole blood may be used as, e.g., when a patient mayalready have lost a significant amount of blood or may already bereceiving a blood transfusion. In such situations, the use ofcrossmatched whole blood in the WBCD introduces the same or similarrisks of serum sickness associated with any transfusion employingcrossmatched whole blood.

The invention also provides kits for preparing an osteogenic wholeblood-derived coagulum device (WBCD) containing one or more BMP-1isoforms for treating a bone defect. For example, in a preferredembodiment, such a kit may be comprised of:

-   -   (a) a vial containing lyophilized BMP-1 isoform(s),    -   (b) a buffer for reconstituting the lyophilized BMP-1        isoforms(s) powder,    -   (c) a syringe and a needle for reconstituting the lyophilized        BMP-1 isoform(s) in the buffer,    -   (d) a vaccutainer for collecting a patient's blood,    -   (e) a sterile solution of 1 M calcium chloride,    -   (f) a fibrin-thrombin mixture,    -   (g) a container for mixing whole blood with the reconstituted        BMP-1 isoform(s) and other ingredients,    -   (h) a spatula or syringe (with or without a needle) (or both)        for applying an osteogenic coagulum to bone ends during open        bone repair surgery, and    -   (i) instructions for the preparation and use of the WBCD        containing BMP-1 isoform(s) using autologous or crossmatched        whole blood.

EXAMPLES Example 1 Purification of BMP-1 Isoform, but not AuthenticOsteogenic BMPs, from Human Blood Plasma by Heparin Sepharose AffinityChromatography, and Protein Identification Using LiquidChromatography-Mass Spectrometry (LC-MS)

This study was originally made to determine whether any osteogenic BMPscould be detected and isolated from human blood plasma.

Plasma Collection

Blood samples from 50 healthy adult humans (21-50 years of age) weredrawn into syringes containing 3.8% sodium citrate to form ananticoagulant-to-blood ratio (v/v) of 1:9. Plasma was obtained bycentrifugation (15 min. at 3000×g), and aliquots of each adult bloodsample were used to make a pooled plasma stock. Aliquot samples werestored at −80° C. prior to analysis.

Affinity Column Purification

Pooled human plasma (80 ml) was diluted 2-fold with 10 mM sodiumphosphate buffer (pH 7), and applied to a 5 ml heparin Sepharose column(Amersham Pharmacia Biotech) previously equilibrated with 10 mM sodiumphosphate buffer (pH 7). Bound proteins were eluted from the column with10 mM sodium phosphate buffer (pH 7) containing 1.0 M and 2.0 M NaCl.

Ammonium Sulfate Precipitation

Saturated ammonium sulfate (SAS) was added into the protein eluatedrop-by-drop with mixing on a vortex to a final concentration of 35%(w/v). Samples were kept on ice for 10 minutes, and centrifuged for 5minutes at 12,000×g. The supernatant was discarded, and the pellet wasprepared for subsequent analysis by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

SDS-PAGE and Western Blot Analysis of the Purified Protein

The pellet was run on standard SDS-PAGE using a 10% gel according to themethod of Laemmli After electrophoresis, one part of the SDS-PAGE gelwas transferred to nitrocellulose and the other was directly stainedwith Coomassie Brilliant Blue (CBB). Nitrocellulose membrane was firstincubated with mouse monoclonal antibody specific for BMP-7 (GeneraResearch Laboratory), and kept overnight at 4° C. Alkalinephosphatase-conjugated goat anti-mouse antibody was used as secondaryantibody for 1 hour at room temperature. The membrane was developed with5 ml of a chromogenic substrate. The other part of the gel was stainedwith Coomassie Brilliant Blue (CBB) under standard staining procedure(0.1% CBB in 45% methanol, 10% acetic acid; 30 minutes at roomtemperature).

The gel was cut into slices corresponding to each protein band asrevealed by staining with CBB. The gel slices were then processed todetermine what proteins were present in each slice using a method ofanalyzing tryptic peptides released from each protein band by HPLC andmass spectrometry (MS) using a nanoelectrospray LC-MS interface asdescribed by Olsen and Mann (Proc. Natl. Acad. Sci. USA, 101:13417-13422 (2004) as modified by Grgurevic et al. (J. Nephrol., 20:311-319 (2007)). Aspects of the steps of this method that arespecifically related to this study are indicated below.

In-Gel Trypsin Digestion Protocol

Bands in the gel were excised from CBB stained gels and digested withtrypsin. Briefly, gel pieces were shrunk with 100 μl of acetonitrile for8 minutes. Liquid was removed and gel pieces were re-swelled with 100 μlof ammonium hydrogencarbonate for 12 minutes and then dried in SpeedVacfor 10 minutes. Dithiothreitol (DTT, 100 μl) was added and incubated for45 minutes at 57° C. Gel pieces were shrunk with 100 μl of acetonitrilefor 8 minutes at 57° C., spun down, and liquid was removed.Iodoacetamide (100 μl) was added to each gel piece and incubated for 45minutes at room temperature in the dark without agitation. Trypsin (10μl) was added per gel piece. Then the gel pieces were spun down andre-swelled for 10 minutes. Samples were incubated overnight at 37° C. ina thermo-mixer.

Peptide Extraction Protocol

Samples were removed from the 37° C. thermo-mixer. A solution (50 μl)containing acetonitrile, water, and formic acid was added. Samples weresonicated for 15 minutes. Supernatant was transferred to the reservetube and 50 μl of acetonitrile were added. Extracts were dried undervacuum in the SpeedVac to complete dryness (about 40 minutes). Peptideswere re-dissolved with 10 μl of solution containing water, methanol, andformic acid. Samples were sonicated for 5 minutes, and stored at −20° C.until analysis.

Mass Spectrometry

Tryptic peptides were analyzed by liquid chromatography-massspectrometry (LC-MS) as follows. Agilent 1100 nanoflow HPLC system(Agilent Technologies, Palo Alto, Calif.) was coupled to a 7-TeslaLTQ-FT mass spectrometer (Thermo Electron, Bremen, Germany) using anano-electrospray LC-MS interface (Proxeon Biosystems, Odense, Denmark).Peptides were separated on a home-made 75 μm C₁₈ HPLC column andmass-analyzed on-the-fly in the positive ion mode. Each measurementcycle consisted of a full mass spectrometry (MS) scan, followed byselected ion monitoring (SIM) scan, MS/MS, and MS/MS/MS scans of thethree most intense ions. This provided a typical peptide mass accuracyof 2 ppm, as well as additional sequence information from the MS/MS andMS/MS/MS fragment ions. Resulting spectra were centroided, and searchedagainst NCBInr database using Mascot search engine (Matrix Science).Searches were done with tryptic specificity, carboxyamidomethylation asfixed modification, and oxidized methionine as variable modification.Mass tolerance of 5 ppm and 0.6 Da was used for MS and MS/MS spectra,respectively.

Results

The LS-MS and immunoblotting analyses revealed twelve (12) trypticpeptides that were compared with the NCBInr database. The 12 peptideswere found not to belong to any known osteogenic BMP, but to the spliceisoform 3 of the precursor of BMP-1-3 (Swiss-Prot: P13497-2; SEQ IDNO:2), i.e., procollagen C-proteinase. The amino acid sequences of eachof the 12 peptides are:

(amino acids 193-203 of SEQ ID NO: 2) GGGPQAISIGK,(amino acids 233-238 of SEQ ID NO: 2) HVSIVR,(amino acids 308-314 of SEQ ID NO: 2) GDIAQAR,(amino acids 352-359 of SEQ ID NO: 2) ISVTPGEK(amino acids 401-411 of SEQ ID NO: 2) LPEPIVSTDSR(amino acids 497-519 of SEQ ID NO: 2) DGHSESSTLIGRYCGYEKPDDIK(amino acids 529-537 of SEQ ID NO: 2) FVSDGSINK,(amino acids 572-584 of SEQ ID NO: 2) CSCDPGYELAPDK,(amino acids 653-660 of SEQ ID NO: 2) SGLTADSK,(amino acids 826-836 of SEQ ID NO: 2) KPEPVLATGSR,(amino acids 841-849 of SEQ ID NO: 2) FYSDNSVQR,(amino acids 958-966 of SEQ ID NO: 2) FHSDDTITK.The 12 peptides had a combined Mascot score of 190, which presents 10⁻¹⁹probability of random (false) identification. No other protein in theNCBInr database matched the same set of peptides. No authenticosteogenic BMP proteins were detected at molecular weight of 100 kDa and35 kDa by LS-MS or by immunoblotting.

The results indicate that authentic osteogenic BMPs do not normallycirculate in the blood of healthy adult humans, whereas BMP-1-3, i.e.,procollagen C-proteinase, is a soluble protein component of normal humanblood.

Example 2 Osteogenic BMP Cannot be Isolated from Human Blood Plasma or24-Hour Urine Rat Sample as Determined by Heparin Sepharose AffinityChromatography and Subsequent Protein Identification Using MassSpectrometry (MS) Plasma Collection

Blood samples from 17 healthy adults (21-50 years) were drawn intosyringes containing 3.8% sodium citrate to form ananticoagulant-to-blood ratio (v/v) of 1:9 Plasma was obtained bycentrifugation (15 mM at 3,000×g), and aliquots of each adult samplewere used to make a pooled plasma stock. Aliquot samples were stored at−80° C. prior to analysis.

Urine Collection

A 24 hour urine sample from healthy rats (Sprague-Dawley, 5 months old,Harlan Winkelmann, Borchen, Germany) was collected in metabolic cages.Prior to purification, the urine was filtrated through Whatmann filterpaper (large pore size) to remove big particles. Samples were stored at−80° C. until studied.

Affinity Column Purification of Plasma Samples

Pooled human plasma (35 ml) was diluted 2-fold with 10 mM sodiumphosphate buffer (pH 7) and applied to a 5 ml heparin Sepharose column(Amersham Pharmacia Biotech), previously equilibrated with 10 mM sodiumphosphate buffer (pH 7). Bound proteins were eluted from the column 10mM sodium phosphate buffer (pH 7) containing 1.0 M and 2.0 M NaCl.

Affinity Column Purification of Urine Rat Samples

A 24 hour urine rat sample (20 ml) was diluted 2-fold with 10 mM sodiumphosphate buffer (pH 7), and applied to a 1 ml heparin Sepharose column(Amersham Pharmacia Biotech), previously equilibrated with 10 mM sodiumphosphate buffer (pH 7). Bound proteins were eluted with 10 mM sodiumphosphate buffer (pH 7) containing 1.0 M and 2.0 M NaCl.

Ammonium Sulfate Precipitation

Saturated ammonium sulfate (SAS) was added into the protein eluatedrop-by-drop on the vortex until the final concentration of 35%. Sampleswere kept on ice for 10 minutes, and centrifuged for 5 minutes at12,000×g. Supernatant was discarded, and pellet was prepared forsubsequent SDS-PAGE analysis. The pellet was run on SDS-PAGE, andproteins in the gel analyzed as described below.

SDS-Polyacrylamide Gel Electrophoresis and Western Blot Analysis of thePurified Protein

The pellet was run on standard SDS-PAGE using a 10% gel according to themethod of Laemmli as described above. After electrophoresis, one part ofthe SDS-PAGE gel was then transferred to nitrocellulose and the otherwas directly stained with CBB. Nitrocellulose membrane was firstincubated with mouse monoclonal antibody specific for BMP-7 (GeneraResearch Laboratory), and kept overnight at 4° C. Alkalinephosphatase-conjugated goat anti-mouse was used as the secondaryantibody for 1 hour at room temperature. The membrane was developed with5 ml chromogenic substrate. The other part of the gel was stained withCBB under standard staining procedure (0.1% CBB in 45% methanol, 10%acetic acid; 30 minutes at room temperature).

The gel was cut into slices corresponding to each protein band asrevealed by staining with CBB. The gel slices were then processed todetermine what proteins were present in each slice using the method ofanalyzing tryptic peptides as described above. Aspects of the steps ofthis method that are specifically related to this study are indicatedbelow.

In-Gel Trypsin Digestion Protocol

Comparing the molecular weight position of bands on the gel stained withCBB with their position on the nitrocellulose membrane, bands 39 kDa, 35kDa, and 50 kDa from the urine sample and bands 39 of kDa and 35 kDafrom plasma sample were excised from CBB stained gel. Gel pieces wereshrunk with 100 μl of acetonitrile for 8 minutes. Liquid was removed andgel pieces were re-swelled with 100 μl of ammonium hydrogencarbonate for12 minutes and then dried in a SpeedVac for 10 minutes. DTT (100 μl) wasadded and incubated for 45 minutes at 57° C. Gel pieces were shrunk with100 μl of acetonitrile for 8 minutes at 57° C., spin down and liquidwere removed. Iodoacetamide (100 μl) was added to each gel piece andincubated for 45 minutes at room temperature in the dark withoutagitation. Trypsin (10 μl) was added per gel piece. Then the pieces werespun down, and re-swelled for 10 minutes. Samples were incubatedovernight at 37° C. in a thermo-mixer.

Peptide Extraction Protocol

Samples were removed from the 37° C. thermo-mixer. A solution (50 μl)containing acetonitrile, water, and formic acid was added. Samples weresonicated for 15 minutes. Supernatant was transferred into the reservetube, and acetonitrile (50 μl) was added. Extracts were dried in theSpeedVac to complete dryness (about 40 min.). Peptides were re-dissolvedwith 10 μl of a solution containing water, methanol, and formic acid.Samples were sonicated for 5 minutes, and stored at −20° C. untilanalysis.

Mass Spectrometry (MS)

Tryptic peptides were analyzed by liquid chromatography-massspectrometry (LC-MS) as follows: Agilent 1100 nanoflow HPLC system(Agilent Technologies, Palo Alto, Calif.) was coupled to a 7-TeslaLTQ-FT mass spectrometer (Thermo Electron, Bremen, Germany) using anano-electrospray LC-MS interface (Proxeon Biosystems, Odense, Denmark).Peptides were separated on a home-made 75 μm C₁₈ HPLC column andmass-analyzed on-the-fly in the positive ion mode. Each measurementcycle consisted of a full MS scan, followed by selected ion monitoring(SIM) scan, MS/MS and MS/MS/MS scans of the three most intense ions.This has resulted in a typical peptide mass accuracy of 2 ppm, as wellas additional sequence information from the MS/MS and MS/MS/MS fragmentions.

Resulting spectra were centroided, and searched against NCBInr databaseusing Mascot search engine (Matrix Science). Searches were done withtryptic specificity, carboxyamidomethylation as fixed modification, andoxidized methionine as variable modification. Mass tolerance of 5 ppmand 0.6 Da was used for MS and MS/MS spectra, respectively.

Results

No authentic, osteogenic BMPs were detected in any of the proteinsisolated from the entire molecular range of purified sera from normalhealthy individuals or from urine of rats by mass spectrometry or byWestern blotting.

Example 3 Lack of Ectopic Bone Formation by Implantation of LyophilizedHuman Blood Samples into Nude Mice and Autologous Rat Lyophilized BloodSamples into Rat Blood Collection

Blood (50 ml) was collected from 10 healthy human individuals. The bloodwas centrifuged to remove cells, and the serum was stored at −20° C.until analyzed. Autologous blood (5 ml) was collected from ten 6-monthsold male Sprague Dawley rats at five time intervals in a period of twoweeks. Samples were centrifuged and the serum was stored at −20° C.until analyzed.

Implantation into Nude Mice and Rats

One bone pellet was formed by mixing 100 mg of human lyophilized bloodwith 200 mg of demineralized rat bone matrix (DBM) and implanted intothe back area of nude mice. In addition, 20 mg of autologous ratlyophilized blood was mixed with 100 mg of DBM and implantedsubcutaneously into the axillar area of the same rats from which theblood had been drawn. Pellets were removed three weeks followingimplantation, fixed and processed for histology.

Results

Tested blood samples implanted under the skin of nude mice were negativefor bone formation, indicating that blood does not contain authenticosteogenic BMPs in an amount that could induce ectopic bone formation inmice and rats.

Example 4 Unlike Recombinant Human BMP-7, Systemically AdministeredBMP-1-1 does not Induce Bone Formation in an Ectopic Bone FormationAssay

Bone pellets consisting of demineralized bone matrix (100 μg) wereimplanted subcutaneously (ectopic site) into 20 adult Sprague Dawleyrats in the axillar region as described previously (Simic et al, J.Biol. Chem., 281:13514 (2006)). Ten rats were then injectedintravenously with 20 μg of recombinant human BMP-7 from days 2 to 7following implantation, while another ten rats were injected on asimilar schedule with recombinant human BMP-1-1. Two weeks followingimplantation, the pellets were removed and processed for histologicalevaluation.

Results

In pellets of rats injected with the BMP-7, cartilage and bone wereformed via a mechanism which involved binding of BMP-7 to the implantedDBM and induction of endochondral bone formation cascade as previouslydescribed (Simic et al, supra). In contrast, in the pellets of ratstreated systemically with BMP-1-1, there was no cartilage or bonedetected, indicating that BMP-1-1 cannot induce bone at an ectopic site.

The results indicate that unlike authentic osteogenic BMP-7,systemically administered BMP-1-1 cannot induce bone formation in anectopic bone formation assay.

Example 5 Cloning and Sequence Analysis of cDNA Encoding BMP-Isoformsfrom Human Placental cDNA Library

The cDNA comprising the coding sequences for BMP-1-1, BMP-1-3, BMP-1-4,and BMP-1-7 were cloned from a human placental cDNA library using theGATEWAY® recombination cloning and expression system (Invitrogen,Carlsbad, Calif.). The correctness of clones was confirmed by standardcolony PCR and restriction enzyme analysis.

The nucleotide base sequences of the cDNA clones were determined and thecorresponding amino acid sequences deduced. The amino acid sequence forthe 83 kDa BMP-1-1 is shown in SEQ ID NO:1. The nucleotide base sequenceof the cDNA clone encoding the BMP-1-3 isoform is shown in SEQ ID NO:3and the corresponding amino acid sequence for the 111 kDa BMP-1-3isoform is shown in SEQ ID NO:2. The amino acid sequence for the 91 kDaBMP-1-7 isoform is shown in SEQ ID NO:7.

The nucleotide base and corresponding amino acid sequences as determinedfor the cDNA clone in this study for the BMP-1-1 and BMP-1-7 isoformswere found to be identical to those present in the EMBL and Swiss-Protdatabases. However, the cDNA sequence for the BMP-1-3 clone asdetermined herein differs at a single nucleotide base from that in theEMBL database. In particular, the EMBL reference sequence (SEQ ID NO:3)has a thymine (T) base at position 1487, whereas the sequence of clonedBMP-1-3 cDNA (SEQ ID NO:5) has an adenine (A), which in turn results ina codon change of a CTG (leucine) in the EMBL sequence to a CAG(glutamine) in the placental BMP-1-3 cDNA sequence isolated by us. Thus,the amino acid sequence of the Swiss-Prot database for BMP-1-3 (SEQ IDNO:2) contains a leucine residue at position 493, whereas the amino acidsequence of the placental BMP-1-3 protein (SEQ ID NO:4) encoded by theisolated cDNA clone contains glutamine at position 493.

Site-directed mutagenesis was performed on the placental BMP-1-3 proteinof the isolated cDNA clone to convert base 1478 of its reported sequence(SEQ ID NO:3), i.e., a switch from adenine (A) to thymine (T). Onexpression, this yielded a “converted” protein of BMP-1-3 having theamino acid sequence of SEQ ID NO: 2.

Results

The placental BMP-1-3 protein, which has the amino acid sequence of SEQID NO:4 when expressed from the library-isolated cDNA clone, and the“converted” BMP-1-3 protein, which has the amino acid sequence asreported in the Swiss-Prot database (SEQ ID NO:2), were both active inprocessing in vitro procollagen type I, II, and III, with the“converted” BMP-1-3 protein being more active at lower concentrations.However, the placental BMP-1-3 expressed from the isolated cDNA cloneprocessed calmodulin and type IV collagen, which properties were notexhibited with the “converted” BMP-1-3 protein. Accordingly, the BMP-1-3isoform expressed from the cloned cDNA of the placental library differsin both amino acid sequence and functional enzymatic properties from theBMP-1-3 protein reported in the Swiss-Prot database.

Example 6 Several Specific BMP-1 Isoforms Circulate in Human Blood inDifferent Diseases Plasma Collection

Blood samples were drawn from 10 healthy adults, from 10 patients eachwho were diagnosed and undergoing treatment for diseases including acutepancreatitis, cirrhosis, acute bone fracture, chronic renal failure ondialysis, and from 4 patients with rare bone diseases, namelyfibrodysplasia ossificians progressive (FOP) and osteogenesis imperfecta(OI). The blood samples were drawn into syringes containing 3.8% sodiumcitrate to form an anticoagulant-to-blood ratio (v/v) of 1:9. Plasma wasobtained by centrifugation (15 minutes at 3000×g), and aliquots of eachblood sample were used to make a pooled plasma stock to represent eachof the listed normal or pathological cases. Aliquot samples were storedat −80° C. prior to analysis.

Affinity Column Purification

80 ml of pooled human plasma from each group of patients was diluted2-fold with 10 mM sodium phosphate buffer (pH 7), and applied to a 5 mlheparin Sepharose column (Amersham Pharmacia Biotech), previouslyequilibrated with 10 mM sodium phosphate buffer (pH 7). Bound proteinswere eluted from the column with 10 mM sodium phosphate buffer (pH 7)containing 1.0 M and 2.0 M NaCl.

Ammonium Sulfate Precipitation

Saturated ammonium sulfate (SAS) was added into the protein eluatedrop-by-drop on the vortex until the final concentration of 35%. Sampleswere kept on ice for 10 minutes, and centrifuged for 5 minutes at12,000×g. Supernatant was discarded, and pellet was prepared forsubsequent SDS-PAGE analysis.

SDS-PAGE and Western Blot Analysis of the Purified Protein

The pellet was run on standard SDS-PAGE on a 10% gel according to themethod of Laemmli After electrophoresis, one part of the SDS-PAGE gelwas then transferred to nitrocellulose and the other was directlystained with Coomassie Brilliant Blue (CBB).

Nitrocellulose membrane was first incubated with rabbit polyclonalantibody specific for the BMP-1 carboxyl terminal domain (Sigma-Aldrich,Chemie GmbH, Germany), and kept overnight at 4° C. Alkalinephosphatase-conjugated anti-rabbit antibody (Invitrogen CorporationCarlsbad, SAD) was used as secondary antibody for 1 hour at roomtemperature. The membrane was developed with 5 ml chromogenic substrate.

The other part of the gel was stained under standard staining procedure(0.1% CBB in 45% methanol, 10% acetic acid; 30 minutes at roomtemperature).

The gel was cut into slices corresponding to each protein band asrevealed by staining with CBB. The gel slices were then processed todetermine what proteins were present in each slice using a method ofanalyzing tryptic peptides as described above. Aspects of the steps ofthis method that are specifically related to this study are indicatedbelow.

In-Gel Trypsin Digestion Protocol

Gel pieces were shrunk with 100 μl of acetonitrile for 8 minutes at 57°C., spun down, and liquid was removed. Gel pieces were re-swelled with100 μl of ammonium hydrogencarbonate for 12 minutes and then dried in aSpeedVac for 10 minutes. DTT (100 μl) was added and incubated for 45minutes at 57° C. Iodoacetamide (100 μl) was added to each gel piece andincubated for 45 minutes at room temperature in the dark withoutagitation. Trypsin (10 μl) was added per gel piece, spun down, and gelpieces were re-swelled for 10 minutes. Samples were incubated overnightat 37° C. on a thermo-mixer.

Peptide Extraction Protocol

Samples were removed from the 37° C. thermo-mixer. A solution (50 μl)containing acetonitrile, water, and formic acid was added. Samples weresonicated for 15 minutes. Supernatant was transferred into the reservetube, and acetonitrile (50 μl) was added. Extracts were dried in theSpeedVac to complete dryness (about 40 minutes). Peptides werere-dissolved with 10 μl of a solution containing water, methanol andformic acid. Samples were sonicated for 5 minutes, and stored at −20° C.until analysis.

Mass Spectrometry

Tryptic peptides were analyzed by liquid chromatography-massspectrometry (LC-MS) as follows: Agilent 1100 nanoflow HPLC system(Agilent Technologies, Palo Alto, Calif.) was coupled to a 7-TeslaLTQ-FT mass spectrometer (Thermo Electron, Bremen, Germany) using anano-electrospray LC-MS interface (Proxeon Biosystems, Odense, Denmark).Peptides were separated on a home-made 75 μm C₁₈ HPLC column andmass-analyzed on-the-fly in the positive ion mode. Each measurementcycle consisted of a full MS scan, followed by selected ion monitoring(SIM) scan, MS/MS and MS/MS/MS scans of the three most intense ions.This resulted in a typical peptide mass accuracy of 2 ppm, as well asadditional sequence information from the MS/MS and MS/MS/MS fragmentions.

Resulting spectra were centroided, and searched against NCBInr databaseusing Mascot search engine (Matrix Science). Searches were done withtryptic specificity, carboxyamidomethylation as fixed modification, andoxidized methionine as variable modification. Mass tolerance of 5 ppmand 0.6 Da was used for MS and MS/MS spectra, respectively.

Results

The results of this study are shown in Table 1 (supra), which providesprofiles of circulating BMP-1 isoforms associated with normal health andthe indicated disorders. The results indicate that the BMP-1-3 isoformis normally present in the blood of healthy individuals but disappearsfrom circulation in patients with acute bone fracture, cirrhosis, andacute pancreatitis. It was surprisingly noted that in FOP and OIpatients BMP-1-3 isoform was still present, but present at more than tentimes the level observed in the blood of healthy individuals.

Disappearance of the BMP-1-3 isoform from the circulation of patientswith acute bone fracture confirms the potential function of BMP-1isoforms in processing the ECM proteins in bone regeneration and repairduring the formation of callus during the rebridgement of fractured boneends. Disappearance of BMP-1-3 from circulation in patients withcirrhosis suggests its involvement in processes related to fibroticchanges in the liver. In acute pancreatitis, several ECM moleculesinvolved in the pathophysiology of the disease eventually require theBMP-1-3 for processing of ECM molecules.

The sera from patients with acute pancreatitis were collected at anearly stage of the disease, i.e., prior to robust serum elevation of thepancreatic enzymes such as pancreatic amylase and lipase. Surprisingly,the blood of these patients contained the BMP-1-7 isoform, which has notbeen previously detected at the protein level.

The BMP-1-5 isoform was found only in patients with chronic kidneyfailure on dialysis, which suggests a specific function for this enzymeisoform, e.g., involvement in the fibrotic processes in bone calledrenal osteodystrophy. Interestingly, this is also the firstdemonstration of BMP-1-5 isoform on the protein level. Previously, theBMP-1-5 isoform was inferred only at the level of tissue mRNAtranscripts.

The presence of BMP-1-3 isoform in circulation was further confirmed byWestern blot using a specific BMP-1-3 antibody developed by Genera (datanot shown).

Example 7 Protection of Kidney Function in Ischemic Acute Renal Failurein Rats by Inhibiting Circulating BMP-1-1 and BMP-1-3 Prior toIschemia/Reperfusion Animals

Female Sprague-Dawley rats weighting about 350-400 g were housed andallowed free access to water and food.

Ischemia/Reperfusion model

Rats were anesthetized with 100 mg/kg ketamine, 10 mg/kg xylazine, and 1mg/kg acepromazine (intramuscularly, im) and placed on a heating tablekept at 37° C. A midline incision was made and both renal pedicles wereclamped for 60 minutes. After removal of the clamp, 5 ml of prewarmednormal saline were instilled into the peritoneal cavity, and theincision was sutured. A total of 24 animals were assigned to twodifferent experimental groups:

-   -   Group 1. Control group (n=12); ischemia/reperfusion model        without therapy (administered physiological saline vehicle, pH        7.2, only)    -   Group 2. Antibody treatment group (n=12); ischemia/reperfusion        model+16 μg of anti-BMP-1-1 antibody (c=1 μg/μl) and 16 μg of        anti-BMP-1-3 antibody (c=1 μg/μl) prior to ischemia/reperfusion        and then for 5 days after ischemia/reperfusion.

Blood samples were obtained before occlusion and at 0, 24, 72, 96, 120,and 168 hours after reperfusion. The plasma was separated bycentrifugation renal function parameters were measured. Rats were killedat day 7 after reperfusion and kidneys were harvested for histologicalanalysis. Therapy was applied in a prophylactic mode at 2 hours prior toclamping and then following the release of the clamps for five daysthereafter.

Assessment of Renal Function

Blood samples (0.5 ml) were obtained from the orbital venous plexus at0, 24, 72, 96, 120, and 168 hours after ischemia. Serum creatinine wasmeasured by Jaffe method (alkaline picrate) and blood urea nitrogen(BUN) by enzymatic glutamate dehydrogenase-UV procedure as previouslydescribed (Vukicevic et al., J. Clin. Invest., 102: 202-214 (1998)). Thecumulative survival rate was observed and recorded for both control andexperimental rats.

Renal Morphology

Kidneys for histological examination were fixed in 2% paraformaldehyde,and 7 μm paraffin sections were cut and stained with haematoxylin andeosin. Tubulointestinal injury, defined as tubular dilatation and/oratrophy, interstitial fibrosis and inflammatory cell infiltrate, as wellas glomerular damage were graded using a semi-quantitative scale from 0to 4 according to the following criteria: 0=no changes; 1=focal changesinvolving 1-25% of the samples; 2=changes affecting 26-50% of thesample; 3=changes involving 51-75% of the sample; and 4=lesionsaffecting more than 75% of the sample as previously described (Vukicevicet al., J. Clin. Invest., id.). Two independent observers performedhistologic studies in a blinded fashion.

Results

Creatinine levels in blood from rats of the untreated control group(Group 1, no antibody therapy) and from rats of the treatment group(Group 2, antibodies against BMP-1-1 and BMP-1-3) are shown in FIG. 1.In control rats, following a 60-minute clamping of both kidneys followedby reperfusion, the creatinine (FIG. 1, diagonal line bars) and BUN (notshown) rose sharply and remained high at 24 hours (1 day) and 72 hours(3 days) following ischemia, then showed normalization at day 7 inanimals that survived the procedure. When antibodies to BMP-1-1 andBMP-1-3 were administered (Group 2) prior to ischemia and then for fivedays following ischemia, both the creatinine (FIG. 1, stippled bars) andBUN (not shown) values remained low. The survival rate was 35% in ratsof the control group (no antibody therapy) and 55% in rats treated withantibodies to BMP-1-1 and BMP-1-3 prior to and followingischemia/reperfusion (data not shown). As observed on the histologyslides (FIGS. 2A-2B), kidneys of rats of the control group that wereexposed to ischemia/reperfusion injury without antibody therapy had lostthe structural integrity in more than 75% of the kidney area withdilated proximal and distal tubules, had lost the tubular epithelium,and about 30% of the entire kidney area was undergoing fibrotic healingdue to necrosis (see, FIG. 2A). In contrast, sections of kidney tissuefrom rats that received antibodies to BMP-1-1 and BMP-1-3 prior toischemia/reperfusion injury indicated significant preservation of kidneystructures (see, FIG. 2B).

These results show that the severity of damage to kidney structure thatwould otherwise occur as the result of an ischemic/reperfusion event canbe prevented by a regimen of systemic administration of neutralizingantibodies to the BMP-1-1 and BMP-1-3 isoforms prior to theischemia/reperfusion event.

Example 8 Enhancing Survival by Systemic Administration of BMP-1 IsoformFollowing Ischemic Acute Renal Failure in Rats Animals

Female Sprague-Dawley rats weighting about 300 g-400 g were housed andallowed free access to water and food.

Ischemia/Reperfusion Model

Rats were anesthetized with 100 mg/kg ketamine, 10 mg/kg xylazine, and 1mg/kg acepromazine (im) and placed on a heating table kept at 37° C. Amidline incision was made, and both renal pedicles were clamped for 60min. After removal of the clamp, 5 ml of normal saline were instilledinto the peritoneal cavity and the incision was sutured. A total of 24animals were assigned to four different experimental groups:

Group 1. Negative control group (n=12); ischemia/reperfusion modelwithout therapy.

Group 2. Positive control group (“BMP-7”) (n=8); 100 μg/kg BMP-7 forfive days.

Group 3. BMP-1-1 treatment group (“BMP-1-1”) (n=8); 4 μg of BMP1-1(c=0.2 μg/μl) for five days.

Group 4. BMP-1-1 antibody treatment group (“BMP-1 Ab”) (n=8); 16 μg ofanti-BMP-1-1 antibody (c=1 μg/μl) for five days after release of clamps(post ischemia/reperfusion event).

Blood samples were obtained before occlusion and at 0, 24, 72, 96, 120,and 168 hours after reperfusion. The plasma was separated bycentrifugation. These samples were used for measurement of renalfunction parameters. Rats were killed at day 7 after reperfusion, andkidneys were harvested for histological analysis. Therapy was appliedfollowing clamping and for five days thereafter.

Assessment of Renal Function

Blood samples (0.5 ml) were obtained from the orbital venous plexus at0, 24, 72, 96, 120, and 168 hours after ischemia. Serum creatinine wasmeasured by Jaffe method (alkaline picrate) and blood urea nitrogen(BUN) by enzymatic glutamate dehydrogenase-UV procedure as previouslydescribed. The cumulative survival rate was observed and recorded forboth control and experimental rats.

Results

Survival of rats in the various treatment groups is shown in FIG. 3. Innegative control rats (Group 1, no therapy) following a 60-minuteclamping of both kidneys followed by a reperfusion, levels of creatinineand BUN rose sharply (not shown), and greater than 60% of the animalsdid not survive (see, FIG. 3, diamond data points). AdministeringBMP-1-1 immediately following reperfusion (“BMP-1-1” group)significantly decreased the mortality and maintained the survival rateat 80% compared to the 40% survival rate of untreated negative controlrats (see, FIG. 3, triangle data points).

Although higher at days 2 and 3 in BMP-1-1 treated rats, serumcreatinine levels sharply declined on day 4 (data not shown), probablydue to a rapid processing of extracellular matrix in the thrombotic areaand a relatively fast recovery of the structural elements that preventedsignificant necrosis due to accumulation of the fibrotic post-necrotictissue. Administration of the BMP-1-1 antibody (“BMP-1 Ab”) for fivedays following the removal of the clamps (see, FIG. 3, cross datapoints) was not effective in preventing a high mortality rate (i.e., aslow as 40% survival rate at day 7 as seen also in the untreated controlgroup).

The results of this experiment indicate that the administration of arecombinant BMP-1 isoform following ischemic acute renal failure iseffective to reduce structural damage to the kidney and to increasesurvival rate of the affected individual.

Example 9 Delaying Progression of Chronic Renal Failure (CRF) in Rats byInhibiting BMP-1 Isoforms Animals

Female Sprague-Dawley rats weighting about 350-400 g were housed andallowed free access to water and food.

5/6 Nephrectomy (Nx) Model of CRF

Rats were anesthetized with 100 mg/kg ketamine, 10 mg/kg xylazine, and 1mg/kg acepromazine (im) and placed on a heating table kept at 37° C. Amidline incision was made, and both renal pedicles were clamped for 60min. The left kidney was removed, and the rats were left for a week torecover. Then, 5/6 of the right kidney mass was removed, and rats wereleft to recover for a period of two weeks. A total of 88 animals wereassigned to 4 different experimental groups:

Group 1. Control group (n=12); 5/6 Nx rats receiving the physiologicalvehicle solution.

Group 2. BMP-1-1 antibody group (n=12); Nx+16 μg of BMP-1-1 antibody(c=1 μg/μl) weekly for a period of 12 weeks

Group 3. BMP-1-3 antibody group (n=12); Nx+16 μg of BMP-1-3 antibody(c=1 μg/μl) weekly for a period of 12 weeks

Group 4. BMP-1-1+BMP-1-3 antibody group (n=12); Nx+16 μg of BMP1-1antibody (c=1 μg/μl) weekly for a period of 12 weeks and 16 μg ofBMP-1-3 antibody (c=1 μg/μl) weekly for a period of 12 weeks.

Blood samples were obtained before surgery and then weekly throughoutthe duration of the experiment. Rats were killed at 12 weeks followingthe removal of the right kidney mass. Therapy was applied intravenously(iv) weekly for a period of 12 weeks.

Assessment of Renal Function

Blood samples (0.5 ml) were obtained from the orbital venous plexusweekly. Serum creatinine was measured by Jaffe method (alkaline picrate)and blood urea nitrogen (BUN) by enzymatic glutamate dehydrogenase-UVprocedure as previously described (Vukicevic et al., J. Clin. Invest.,op. cit.). The cumulative survival rate was observed and recorded forboth control and experimental rats.

Renal Morphology

Kidneys for histological examination were fixed in 2% paraformaldehyde,and 7 μm paraffin sections were cut and stained with haematoxylin andeosin. Kidney damage was graded as described (Borovecki et al., in Bonemorphogenetic proteins—Bone regeneration and beyond, edited by VukicevicS. and Sampath K. T., 2002). Briefly, the structure of glomeruli, kidneytubules, and the amount of interstitial fibrosis were measured on thekidney area using an automated computer program. The measured parameterswere expressed as a number of vital versus damaged glomeruli and as apercent of fibrotically altered kidney area. Two independent observersperformed histologic studies in a blinded fashion.

Results

Following 12 weeks of therapy, control rats (Group 1), which receivedonly the vehicle solution, had creatinine values above 300 mEq/L Animalstreated with a single antibody, i.e., antibody to BMP-1-1 (Group 2) orantibody to BMP-1-3 (Group 3), or with a combination of both antibodies(Group 4) had significantly lower creatinine serum values as compared tocontrol rats. In particular, rats treated with anti-BMP-1-1 antibody(Group 2) or with anti-BMP-1-3 antibody (Group 3) had, respectively, 36%and 39% lower creatinine serum values than control rats. Creatinineserum values were 54% lower in rats treated with a combination of bothanti-BMP1-1 and anti-BMP-1-3 antibodies than in the control rats. Inanimals treated with a combination of both antibodies (Group 4), thefibrotic area was reduced by 57% relative to control rats, while in ratstreated with only the anti-BMP-1-1 antibody (Group 2) or with only theanti-BMP-1-3 antibody (Group 3), the fibrotic area was reduce by 23% and16%, respectively. In addition, the fibrotic area was reduced by 43% inrats treated with a combination of both antibodies as compared to ratstreated with BMP-7, a positive control.

These results indicate that inhibition of BMP-1-1 and BMP-1-3 in a modelof a chronic renal failure (CRF) delayed the progression of the diseaseby maintaining the structural integrity of glomeruli and preventingaccumulation of fibrotic tissues, thus, improving the kidney function byabout 50% in a period of 12 weeks following CRF. This relates toincreasing a human life span by about 120 months or about 10 years.

Example 10 Acceleration of Fracture Repair with SystemicallyAdministered BMP-1-1 and Localization of BMP-1-1 at Orthotopic Site ofBone Fraction Animals and Experimental Protocol

Fifty (50) 4-month old Sprague-Dawley female rats were used in thisstudy. Animals weighed approximately 300 grams (g). They were kept instandard conditions (24° C., 12 hour/12 hour light/dark cycle) in20×32×20 cm cages during the study and were allowed free access to waterand pelleted commercial diet (Harlan Teklad, Borchen, Germany). Ratswere divided into three treatment groups and two control groups:

Group 1. Control rats (10) were treated with a Kirschner wire followingsurgically produced fracture and then treated systemically with avehicle solution (physiological saline, pH 7.2) only.

Group 2. Rats treated with BMP-1-1 (10 μg/kg) for a period of one week.Ten rats were treated with Kirschner wire following fracture of thefemur and then intravenously treated with BMP-1-1.

Group 3. Rats treated with BMP-1-1 (10 μg/kg) for a period of threeweeks. Ten rats were treated with Kirschner wire following fracture ofthe femur and then intravenously treated with BMP-1-1.

Group 4: Rats treated with BMP-1-1 (10 μg/kg) for a period of fiveweeks. Ten rats were treated with Kirschner wire following fracture ofthe femur and then intravenously treated with BMP-1-1.

Group 5: Positive control. Ten rats were treated with a Kirschner wirefollowing fracture of the femur and then injected systemically with 100μg/kg of BMP-7 for a period of five (5) weeks.

Anesthetized rats were prepared for surgery by shaving and cleaning thelower extremities. With a medial peripatellar incision, the patella wasdislocated laterally exposing the femoral condyle. A Kirschner wire (1.1mm in diameter and 2.7 cm long) was introduced into the intramedullarycanal through the intercondylar notch. The Kirschner wire did notprotrude into the knee joint or interfere with motion of the patella.After closing the knee joint, the mid-diaphysis of the pinned rightfemur was fractured by applying a bending force, as described byBonarens and Einhom (J. Orthop. Res., 97:101 (1984)). Radiographs wereobtained immediately after surgery, and rats with proximal or distalfractures were excluded from this experiment so that only mid-diaphysealfractures were included in this study.

All animals were sacrificed following seven weeks of therapy.Radiographs were taken at week one and seven following surgery in twoplanes: AP (anterior-posterior) and LL (latero-lateral).

Biodistribution and Pharmacokinetics of ¹²⁵I-Labeled BMP-1-1(¹²⁵I-BMP-1-1)

Recombinant human BMP-1-1 was radioiodinated with 5 mCi of carrier-freeNa ¹²⁵I using a modification of the lactoperoxidase method. Gelfiltration on a Sephadex G-25 column was used to separate radioiodinatedBMP-1-1 (¹²⁵I-BMP-1-1) from the free iodide. The column was eluted with20 mM sodium acetate buffer, pH 4.5 containing 0.2% Tween-80 and 0.1%ovalbumin. The specific activity of the ¹²⁵I-BMP-1-1 preparation used inthis study was 0.273 mCi/mg. Rats (n=10) received a single injection of¹²⁵I-BMP-1-1 at a dose level of 10 μg/kg with the activity of 20 μCi.Injection volume was 500 μl Animals were sacrificed 30 minutes, 1, 3, 6and 24 hours following injection. Tissues were removed, weighed, andradioactivity was measured in a gamma counter. The relative uptake of1251-BMP-1 by tissues during time was expressed as nanograms (ng) of¹²⁵I-BMP-1 per gram (g) wet tissue weight. The experiments were alsoperformed in five rats with acutely fractured femurs on day fivefollowing surgical osteotomy of the femur.

In Vivo and Ex Vivo Bone Mineral Density (BMD) Measurement by DXA

At two-week intervals (in period of 10 weeks), the animals were scannedfor bone density measurements by dual-energy X-ray absorptiometry (DXA;Hologic QDR-4000, Hologic, Waltham, Mass.). At the end of theexperiment, animals were anesthetized, weighed, and euthanized. Theright femur was removed and fixed in 70% ethanol and was used fordetermination of the bone mineral content (BMC) and BMD by DXA equippedwith Regional High Resolution Scan software. The scan field size was5.08×1.902 cm, resolution was 0.0254×0.0127 cm, and the speed was 7.25mm/s. The scan images were analyzed and the bone area, bone mineralcontent, and bone density of whole bone.

PQCT

Isolated femurs were scanned by a peripheral quantitative computerizedtomography (PQCT) X-ray machine (Stratec XCT Research M; Norland MedicalSystems, Fort Atkinson, Wis.) with software version 5.40. Volumetriccontent, density, and area of the total bone, trabecular, and corticalregions were determined.

MicroCT

The microcomputerized tomography (MicroCT) apparatus (p CT 40) and theanalyzing software used in these experiments were obtained from SCANCOMedical AG (Bassersdorf, Switzerland). The right femur was scanned in250 slices, each 13 μm thick in the dorsoventral direction.Three-dimensional reconstruction of bone was performed using thetriangulation algorithm. The trabecular bone volume (BV, mm³),trabecular number (Tb. N, 1/mm), the trabecular thickness (Tb. Th, μm),and the trabecular separation (Tb. Sp, μm) were directly measured on3-dimensional (3D) images using the method described by Hildebrand etal. (Comp. Meth. Biochem. Biomed. Eng., 1: 15 (1999)). The trabecularbone pattern factor (TBPf) and the structure model index (SMI) werecomputed using software provided with the microCT machine.

Histology

The femur was removed for histologic analyses, embedded in paraffin, cutin 10 μm thick sections, stained with hemalaun-eosin and toluidine blue.

Results

Radioactively labeled BMP-1-1 was injected intravenously into healthyrats and into rats with fractured femurs. In healthy animals,radioactive BMP-1-1 accumulated predominantly in the liver (23%), bones(31%), and muscles (9%). In rats with a fracture, 80% of injectedBMP-1-1 accumulated at the fracture site.

Rats treated with BMP-1-1 for one week with daily intravenous injectionsshowed 43% accelerated bone regeneration, which was calculated based ona scoring system of bone repair as previously described (Paralkar etal., Proc. Natl. Acad. Sci. USA, 100: 6736 (2003)). The formed calluswas bigger by 43% in rats treated with BMP-1 for one week, and it wasincreased by 63% and 71% in rats treated with BMP-1 for three to fiveweeks, respectively. The bone healing was accelerated by 40-80% in ratstreated with BMP-1-1 for a period of one or five weeks, respectively, asevidenced by full rebridgement of the three or four cortices of ratfemurs.

In vivo bone mineral density measurement showed increased accumulationof mineral in the formed callus, while PQCT analyses showed increasedmineral accumulation on the cortical bone of fractured femurs. MicroCTmeasurement showed increased accumulation of newly formed trabeculi inthe regenerating fracture at seven weeks following surgical osteotomy.

These results of this study of acute femur fracture in rats collectivelyindicate that the vast majority (e.g., about 80%) of systemicallyadministered BMP-1-1 becomes localized in the orthotopic site of a bonefracture and that systemically administered BMP-1-1 is effective ataccelerating healing of such acute fractured femurs.

Example 11 Systemically Administered BMP-1-1 into Rats with FracturedFemur

Employing similar procedures as in Example 10, above, a study was madeto compare the effect of systemic administration of BMP-1-1 isoform,BMP-7, and antibody to the BMP-1-1 isoform on healing of fracturedfemurs in rats.

At 4 weeks following fracture, the callus at the fracture site in ratstreated systemically with BMP-1 isoform was about 20% bigger than thatin untreated control rats and about 90% bigger than in rats treatedsystemically with BMP-7.

Results at 8 weeks following fracture are shown in FIGS. 4A-4F. The areaof the fracture is encircled in each of the pictured femurs FIGS. 4A-4F.Systemic administration of BMP-1-1 to rats with a fractured femurresulted in accelerated healing as compared to systemic administrationof BMP-7. The fracture line had almost disappeared, and the corticalbone had rebridged in rats treated systemically with BMP-1-1 (see, bonesin FIG. 4A and FIG. 4D), whereas the fracture line was still visible inrats treated systemically with BMP-7 (see, bones in FIGS. 4B, 4C, and4E). Systemic administration of neutralizing antibody to BMP-1-1 delayedfracture healing (see, bone in FIG. 4F).

The results indicate that systemic administration of a BMP-1 isoform isan effective method for treating bone defects.

Example 12 Locally Administered BMP-1-1 into Rats with Fractured FemurAnimal Model of Fracture

Twenty four (24) 3-month old Sprague-Dawley male rats (350 g) weretreated with Kirschner wire following fracture of the femur. Rats weredivided into the following three treatment groups:

Group 1. Control rats (8) were treated with a whole (autologous)blood-derived coagulum device containing vehicle solution only(physiological solution; no BMP-1-1, no BMP-7).

Group 2. Rats treated locally with whole blood-derived coagulum devicecontaining BMP-1 (10 μg/kg of BMP-1-1).

Group 3. Rats (8) treated with whole blood-derived coagulum devicecontaining BMP-7 (10 μg/kg).

All animals were sacrificed seven weeks after surgery. Radiographs weretaken at week 1, 4, and 7 in two planes, i.e., AP (anterior-posterior)and LL (latero-lateral).

Anesthetized rats were prepared for surgery by shaving and cleaning thelower extremities. With a medial peripatellar incision, the patella wasdislocated laterally exposing the femoral condyle. A Kirschner wire (1.1mm in diameter and 2.7 cm long) was introduced into the intramedullarycanal through the intercondylar notch. The Kirschner wire did notprotrude into the knee joint or interfere with motion of the patella.After closing the knee joint, the mid-diaphysis of the pinned rightfemur was fractured by applying a bending force, as described byBonarens and Einhom (J. Orthop. Res., 97: 101 (1984)). Radiographs wereobtained immediately after surgery, and rats with proximal or distalfractures were excluded from this experiment, so that the onlymid-diaphyseal fractures were included in this study.

Preparation of Whole Blood-Derived Coagulum Device (WBCD) ContainingBMP-1

Whole blood-derived coagulum devices (WBCDs) for treating bone fractureswere prepared to treat bone fractures in rat femurs. Briefly, 1 ml ofautologous whole blood was drawn from the orbital plexus of each rat.The whole blood was then combined with a thrombin-fibrin reagent, 1 Mexogenous calcium chloride, and the indicated amount of BMP-1-1 orBMP-7, and then incubated at room temperature for 30-45 minutes topermit coagulum formation prior to implantation into the fractured femurof the rat that provided the corresponding autologous blood.

Biomechanical Testing

Femurs from both sides were removed for biomechanical testing, whichincluded three-point bending as previously described (Simic et al., J.Biol. Chem., 281: 13472 (2006)). The healthy bones from thecontra-lateral side were used as positive controls. Both three-pointbending test and the indentation test were used for measuringbiomechanical characteristics of both the cortical and the trabecularbone.

Results

Radiographic analysis of X-rays showed that in rats treated with a WBCDcontaining only the vehicle solution (no BMP-1-1, no BMP-7) as a controlat 4 weeks following surgery, 0.6±0.03 cortices healed, while at sevenweeks following surgery 1.8±0.4 cortices healed. The callus area was24.3±7.8 mm² at four weeks and 18.7±6 4 mm² at seven weeks.

In rats treated with a whole blood-derived coagulum device+BMP-1-1 atfour weeks 1.3±0.5 (t-test, P>0.01 vs control) cortices healed, while atseven weeks 2.9±0.9 (t-test, P>0.01) cortices healed. The callus areawas 13.4±4.7 mm² (t-test, P>0.01 vs control), and at seven weeks it was7.6±3.8 mm² (t-test, P>0.05 vs control).

In rats treated with WBCD+BMP-7 at four weeks 1.7±0.7 (t-test, P>0.01 vscontrol and P>0.1 vs BMP-1) cortices healed, while at seven weeks3.2±1.4 (t-test, P>0.01 vs control and P>0.1 vs BMP-1) cortices healed.The callus area was 11.3±3.9 mm² (t-test, P>0.01 vs control and P>0.1 vsBMP-1), and at seven weeks it was 6.7±2.9 mm² (t-test, P>0.05 vs controland P>0.1 vs BMP-1).

These results indicate that locally administered BMP-1-1 at anorthotopic site (defect site) in a model of femoral fracture repairsignificantly accelerated the bone fracture healing as compared tocontrol rats. Surprisingly, when BMP-7 was used in a composition withWBCD, femurs healed faster than in control rats, but were not differentfrom animals treated with BMP-1-1, which is an ECM processing enzyme.BMP-7 is commercially used with bovine collagen as a carrier. Bovinecollagen implanted alone in a similar model of bone repair in a ratinhibits bone repair as compared to untreated control rats.

Biomechanical Testing

Three point bending test indicated that BMP-1-1 treated femurs needed asignificantly greater maximal load to re-fracture as compared to controlfemurs treated only with the whole blood-derived coagulum device (noBMP-1-1) (see, Table 2, below). As compared with the femur from theopposite leg (contralateral femur), bones treated with BMP-1-1 required26% less load to cause re-fracture; whereas control bones needed 51%less load to re-fracture than the normal contralateral bones (see Table2).

The maximal load needed to break BMP-7 treated bones was notstatistically different from those treated with BMP-1-1 (see, Table 2,below). These results confirmed the radiographic findings collectivelyindicating that BMP-1-1 accelerates bone repair and regeneration ofacute fractures in a rat model, and that it is equally as effective asBMP-7 when used with the whole blood-derived coagulum device.Indentation test of trabecular bone indicates that BMP-1-1 treated boneshad more trabecular bone than control animals (see, Table 3).

TABLE 2 Results of three point bending test on rat femurs after therapyBMP-1 BMP-7 Parameter Control BMP-1-1 BMP-7 contralateral contralateralFμ (N) 119.99 ± 19.77  175.32 ± 24.87*  189.12 ± 28.69* 212.33 ± 37.82234.56 ± 24.59 S (N/mm) 266.84 ± 48.81 356.12 ± 53.09 377.40 ± 39.94390.27 ± 43.30 402.75 ± 40.13 W (mJ)  91.67 ± 23.35 106.08 ± 15.54116.06 ± 17.80 122.25 ± 18.16 131.15 ± 32.65 T (MJ/m³)  8.65 ± 2.4911.84 ± 1.7  11.33 ± 1.5  12.12 ± 1.61 12.36 ± 3.89 *P < 0.01 vscontrol, one way ANOVA-Dunnett test

TABLE 3 Results of indentation test on rat femurs after therapy BMP-1BMP-7 Parameter Control BMP-1-1 BMP-7 contralateral contralateral Fμ (N)67.47 ± 25.7 84.30 ± 13*  104.95 ± 31*   101.31 ± 32.73 129.13 ± 19.5* S(N/mm)  93.25 ± 44.33 118.03 ± 14.34 132.11 ± 32.68* 180.36 ± 38.6*170.54 ± 32.6* W (mJ) 54.62 ± 14.2  83.89 ± 15.1* 93.65 ± 16.5* 104.21 ±25.2* 106.24 ± 16.8  σ (N/mm²) 21.49 ± 11.3 31.37 ± 1.19 43.68 ± 9.8*  51.61 ± 10.42* 59.28 ± 6.2* *P < 0.01 vs control, one way ANOVA-Dunnetttest

Example 13 The Release of BMP-4 and BMP-7 into the Medium of In VitroCultured Rat Calvariae Explant Cultures Treated with BMP-1-1 and BMP-1-3

Rat fetuses that were 18 days old were obtained from pregnant rats andtheir calvariae were isolated, cleaned, equally sized, and placed intocultures containing bone specific medium as previously described(Vukicevic et al., Proc. Natl. Acad. Sci. USA, 86: 8793 (1989)). Suchcalvariae explant cultures produce bone cells as well as extracellularmatrix (ECM). At 48 hours following culture, the explanted calvariaewere treated with 100 ng/ml BMP-1-1 or 100 ng/ml BMP-1-3 daily for aperiod of 3 days. The medium was collected daily, stored at −20° C., andon day 4 purified over a heparin column. Following purification over aheparin column, the protein concentration was determined and BMP-2,BMP-4, BMP-6, and BMP-7 were detected by immunoblotting as previouslydescribed (Simic et al., J. Biol. Chem., 286: 13472 (2006)).

The results indicated that in the medium of control cultures there wereno detectable amounts of authentic osteogenic BMPs found, while in themedium of calvariae treated with BMP-1-1, the mature domain of BMP-4 wasdetected, whereas BMP-2, BMP-6 and BMP-7 were not detected. Theseresults indicate that BMP-1-1 has an effect on the release of BMP-4 fromculture explants consisting of fetal calvariae rich in bone cells andECM, which appears to act as a repository of stored authentic BMPmolecules (see, also, Martinovic et al., Arch. Cytol. Histol., 1: 23(2006)). In the medium of cultures treated with BMP-1-3 in addition toBMP-4, BMP-7 was detected, indicating that BMP-1-3 releases moreauthentic BMPs from ECM than BMP-1-1.

Example 14 Synergistic Acceleration of Bone Defect Healing in RabbitsTreated Locally with BMP-1-1 and BMP-7 Animals

An ulnar segmental-defect model was used to evaluate bone healing inadult male New Zealand White rabbits (3 kg to 4 kg in weight). Theimplants consisted of blood coagulum as a carrier to which differentamounts of recombinant human BMP-1-1 and recombinant human mature BMP-7were added (Genera Research Laboratory). These animals were comparedwith animals receiving blood coagulum implant alone (negative control).Rabbits were treated with anti-parasitics one week before surgeryAnimals were also given enrofloxacin, by intramuscular injection, at oneday before operation and then ten days following surgery.

With the rabbit under anesthesia and analgesia, one forelimb was shavedand then prepared and draped in a sterile fashion. A lateral incision,approximately 2.5 centimeters in length, was made, and the tissuesoverlying the ulna were dissected. A 1.5-centimeter segmentalosteoperiostal defect was created in the middle of the ulna with anoscillating saw. The radius was left intact for mechanical stability,and no internal or external fixation devices were used. After copiousirrigation with saline solution to remove bone debris and spilled marrowcells, the implant was packed carefully into place to fill the defect.Coagulum was then overlaid with serum. The soft tissues were closedmeticulously in layers to contain the implant. The animals were allowedfull weight-bearing activity, water, and rabbit chow.

WBCD Preparation

Blood samples were collected from rabbit marginal ear veins into tubeswithout any anticoagulants substance in a volume of 1.5 mL, one daybefore surgery. BMP-1-1 and BMP-7 were added into blood in amounts of 14μg and 100 μg, respectively. Blood samples were left at 4° C. tocoagulate. The next day, samples were centrifuged at 8000×g for 5minutes. Liquid part (serum) was removed and saved, and coagulum wasready to use.

The rabbits were divided into one of the groups listed below and defectshave been treated as follows:

Group 1. Control rabbits treated with the whole blood coagulum device(WBCD) without BMP or BMP-1 isoform only (n=8).

Group 2. Rabbits treated with WB CD containing 14 μg/1.5 mL of BMP-1-1.

Group 3. Rabbits treated with WB CD containing 100 μg/1.5 mL of BMP-7.

Group 4. Rabbits treated with WBCD containing 14 μg/1.5 mL of BMP-1+100μg of BMP-7/1.5 mL.

Results

The results are shown in FIGS. 5-8. Rabbit ulna defects did not heal inthe control rabbits (Group 1) treated with WBCD only (no BMP-1-1, noBMP-7), as observed by X-ray biweekly follow up. The unhealed defect ina representative bone after 6 weeks from the control group is shown inFIGS. 5A and 5B (two views of the same bone).

Results after 6 weeks in a representative bone from rabbits treatedlocally with a WBCD having BMP-1-1 (Group 2) are shown in FIGS. 6A and6B. Results after 6 weeks in a representative bone from rabbits treatedlocally with WBCD having BMP-7 (Group 3) are shown in FIGS. 7A and 7B.Results after 6 weeks in a representative bone from rabbits treatedlocally with WBCD having BMP-1-1 and BMP-7 (Group 3) are shown in FIGS.8A and 8B. Rabbits treated with BMP-7-containing WBCD (Group 3)rebridged the bone defect at 8 weeks following surgery, while rabbitstreated with BMP-1-1-containing WBCD (Group 2) showed initial boneformation as early as two weeks and advanced healing at 8 weeksfollowing surgery. However, rabbits treated locally with a WBCD having acombination of both BMP-1-1 and BMP-7 (Group 4), had a synergistichealing of the ulnar defect with a complete rebridgmenet of the defectand formation of the new cortex with a pronounced remodelling of newlyformed bone as early as 6 weeks (see, FIGS. 8A and 8B).

These results indicate that BMP-1-1 and BMP-7 applied locally at anorthotopic site of a fracture act synergistically to accelerate boneregeneration.

All patents, applications, and publications cited in the above text areincorporated herein by reference.

Other variations and embodiments of the invention described herein willnow be apparent to those of skill in the art without departing from thedisclosure of the invention or the claims below.

1. A method of treating ischemic acute renal failure in an individualcomprising administering a BMP-1 isoform systemically to the individualafter diagnosis of renal injury.
 2. A method of treating an individualfor a defect or disorder in bone or soft tissue of an individualcomprising: (a) diagnosing a defect or disorder in a bone or soft tissuein an individual by steps comprising: (i) determining the profile ofBMP-1 isoforms in the blood and (ii) comparing the profile to a standardblood profile of BMP-1 isoforms associated with various defects anddisorders, (b) administering to the individual an amount of at least oneBMP-1 isoform effective to enhance the therapeutic effect of anosteogenic BMP toward the diagnosed defect or disorder, or administeringto the individual an amount of one or more antibody molecules specificfor one or more BMP-1 isoforms effective to inhibit the effects of saidone or more BMP-1 isoforms in the progression of the diagnosed defect ordisorder.
 3. The method according to claim 2, wherein said standardblood profile of BMP-1 isoforms is Table
 1. 4. The method according toclaim 2, wherein said soft tissue is in a parenchyma organ.
 5. Themethod according to claim 4, wherein said parenchyma organ is selectedfrom the group consisting of liver, pancreas, and kidney.
 6. The methodaccording to claim 2, wherein in step (b) a BMP-1 isoform isadministered to the individual systemically or locally at a site of thedefect or disorder.
 7. The method according to claim 2, wherein in step(b) an antibody to a BMP-1 isoform is administered systemically.
 8. Themethod according to claim 2, wherein said BMP-1 isoform or said antibodymolecule is administered systemically via intravenous or intramuscularinjection.
 9. A method of pretreating an individual to resolve clotsthat may occur during thoracic or abdominal surgery comprisingadministering a BMP-1 isoform to the individual prior to surgery in anamount effective to inhibit the occurrence of clots.
 10. A method oftreating acute pancreatitis in an individual comprising administering tothe individual a therapeutically effective amount of at least oneantibody molecule specific for a BMP-1 isoform.
 11. The method accordingto claim 10, wherein the antibody molecule is an anti-BMP-1-7 antibodymolecule.
 12. A method of treating pancreatitis in an individualcomprising administering to an individual suffering from pancreatitis,after the acute phase of the inflammatory process, an amount of a BMP-1isoform in an amount effective to promote pancreatic regeneration. 13.The method according to claim 12, wherein the BMP-1 isoform administeredis BMP-1-7.
 14. An osteogenic whole blood-derived coagulum device (WBCD)for treating a bone defect in an individual prepared by the stepscomprising: (a) preparing a mixture comprising: (1) whole blood, (2) aBMP-1 isoform, (3) an exogenous substance supplying calcium ions, and(4) optionally, a mixture of fibrin and thrombin, and (5) optionally, anosteogenic BMP; (b) incubating the mixture of step (a) until amechanically stable gel is formed, wherein said mechanically stable gelis a homogenous, cohesive, syringeable, injectable, and malleablecoagulum gel.
 15. The osteogenic WBCD according to claim 14, wherein thewhole blood is autologous whole blood drawn from the individual or iswhole blood that has been crossmatched with the individual.
 16. Theosteogenic WBCD according to claim 14, wherein step (a) is carried outby first combining the fibrin-thrombin mixture, calcium ion substance,and the BMP-1 isoform to form a first mixture, adding whole blood to thefirst mixture to form a second mixture, and incubating the secondmixture until a mechanically stable gel is formed.
 17. A method oftreating a bone defect in an individual comprising administering theWBCD according to claim 14 to the bone defect.
 18. The method accordingto claim 17, wherein the WBCD is administered by implantation or byinjection into the bone defect.
 19. A kit for preparing an osteogenicwhole blood-derived coagulum device (WBCD) comprising: (a) a vialcontaining one or more lyophilized BMP-1 isoform, (b) a buffer forreconstituting the lyophilized BMP-1 isoforms(s), (c) a syringe forreconstituting the lyophilized BMP-1 isoform(s) in the buffer, (d) avaccutainer for collecting a patient's blood, (e) a sterile solution of1 M calcium chloride, (f) a fibrin-thrombin mixture, (g) a container formixing whole blood with the reconstituted BMP-1 isoform(s) and otheringredients, (h) a spatula or syringe suitable for applying anosteogenic coagulum to bone ends during open bone repair surgery, and(i) instructions for the preparation and use of a WBCD comprised ofwhole blood mixed with one or more BMP-1 isoforms, calcium chloride and,optionally, a mixture comprising fibrin and thrombin, to form amechanically stable gel suitable for application to a bone defect,wherein said mechanically stable gel is a homogenous, cohesive,syringeable, injectable, and malleable coagulum gel.
 20. An isolatedBMP-1 isoform having the amino acid sequence of SEQ ID NO:4.
 21. Anisolated polynucleotide encoding the polypeptide having the amino acidsequence of SEQ ID NO:4.
 22. The polynucleotide according to claim 21having the nucleotide sequence of SEQ ID NO:5.